Streptococcus pyogenes antigens and corresponding dna fragments

ABSTRACT

The present invention relates to antigens, more particularly antigens of  Streptococcus pyogenes  (also called group A Streptococcus (GAS)) bacterial pathogen which are useful as vaccine component for therapy and/or prophylaxis.

FIELD OF THE INVENTION

[0001] The present invention is related to antigens, more particularlyBVH-P2, BVH-P3, BVH-P4, BVH-P5, and BVH-P6 antigens of Group AStreptococcus (S. pyogenes) bacterial pathogen which may be used toprevent, diagnose and/or treat streptococcal infections.

BACKGROUND OF THE INVENTION

[0002] Streptococci are gram (+) bacteria which are differentiated bygroup specific carbohydrate antigens A through 0 which are found at thecell surface. S. pyogenes isolates are further distinguished bytype-specific M protein antigens. M proteins are important virulencefactors which are highly variable both in molecular weights and insequences. Indeed, more than 80-M protein types have been identified onthe basis of antigenic differences.

[0003]S. pyogenes is responsible for many diverse infection types,including pharyngitis, erysipelas and impetigo, scarlet fever, andinvasive diseases such as bacteremia and necrotizing fasciitis. Aresurgence of invasive disease in recent years has been documented inmany countries, including those in North America and Europe. Althoughthe organism is sensitive to antibiotics, the high attack rate and rapidonset of sepsis results in high morbidity and mortality.

[0004] To develop a vaccine that will protect hosts from S. pyogenesinfection, efforts have focused on virulence factors such as thetype-specific M proteins. However, the amino-terminal portion of Mproteins was found to induce cross-reactive antibodies which reactedwith human myocardium, tropomyosin, myosin, and vimentin, which might beimplicated in autoimmune diseases. Others have used recombinanttechniques to produce complex hybrid proteins containing amino-terminalpeptides of M proteins from different serotypes. However, a safe vaccinecontaining all S. pyogenes serotypes will be highly complex to produceand standardize.

[0005] In addition to the serotype-specific antigens, other S. pyogenesproteins have generated interest as potential vaccine candidates. TheC5a peptidase, which is expressed by at least S. pyogenes 40 serotypes,was shown to be immunogenic in mice, but its capacity to reduce thelevel of nasopharyngeal colonization was limited. Other investigatorshave also focused on the streptococcal pyrogenic exotoxins which appearto play an important role in pathogenesis of infection. Immunizationwith these proteins prevented the deadly symptoms of toxic shock, butdid not prevent colonization.

[0006] The University of Oklahoma has set up a genome sequencing projectfor S. pyogenes strain M1 GAS (http://dnal.chem.ou.edu/strep.html).

[0007] Therefore there remains an unmet need for S. pyogenes antigensthat may be used vaccine components for the prophylaxis and/or therapyof S. pyogenes infection.

SUMMARY OF THE INVENTION

[0008] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 70%identity to a second polypeptide comprising a sequence chosen from SEQID Nos: 2,4,6,8,10,12,14 and 16 or fragments or analogs thereof.

[0009] According to one aspect, the present invention relates topolypeptides which comprise an amino acid sequence chosen from SEQ IDNos: 2,4,6,8,10,12,14 and 16 or fragments or analogs thereof.

[0010] In other aspects, there are provided polypeptides encoded bypolynucleotides of the invention, pharmaceutical compositions, vectorscomprising polynucleotides of the invention operably linked to anexpression control region, as well as host cells transfected with saidvectors and methods of producing polypeptides comprising culturing saidhost cells under conditions suitable for expression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In FIGS. 1, 3, 5, 7, 9, the underlined portion of the sequencerepresents the region coding for the leader peptide. In FIGS. 2, 4, 6,8, 10, the underlined portion of the sequence represents the leaderpeptide.

[0012]FIG. 1 represents the DNA sequence of BVH-P2 gene from serotype M3S. pyogenes strain ATCC12384; SEQ ID NO: 1.

[0013]FIG. 2 represents the amino acid sequence BVH-P2 polypeptide fromserotype 3 S. pyogenes strain ATCC12384; SEQ ID NO: 2.

[0014]FIG. 3 represents the DNA sequence of BVH-P3 gene from serotype M1S. pyogenes strain ATCC700294; SEQ ID NO: 3.

[0015]FIG. 4 represents the amino acid sequence BVH-P3 polypeptide fromserotype M1 S. pyogenes strain ATCC700294; SEQ ID NO: 4.

[0016]FIG. 5 represents the DNA sequence of BVH-P4 gene from serotype M1S. pyogenes strain ATCC700294; SEQ ID NO: 5.

[0017]FIG. 6 represents the amino acid sequence BVH-P4 polypeptide fromserotype M1 S. pyogenes strain ATCC700294; SEQ ID NO: 6.

[0018]FIG. 7 represents the DNA sequence of BVH-5 gene from serotype M1S. pyogenes strain ATCC700294; SEQ ID NO: 7.

[0019]FIG. 8 represents the amino acid sequence BVH-P5 polypeptide fromserotype M1 S. pyogenes strain ATCC700294; SEQ ID NO: 8.

[0020]FIG. 9 represents the DNA sequence of BVH-P6 gene from serotype M1S. pyogenes strain ATCC700294; SEQ ID NO: 9.

[0021]FIG. 10 represents the amino acid sequence BVH-P6 polypeptide fromserotype M1 S. pyogenes strain ATCC700294; SEQ ID NO: 10.

[0022]FIG. 11 represents the DNA sequence of BVH-P4 gene from serotypeM3 S. pyogenes strain ATCC123834; SEQ ID NO: 11.

[0023]FIG. 12 represents the amino acid sequence BVH-P4 polypeptide fromserotype M3 S. pyogenes strain ATCC12384; SEQ ID NO: 12.

[0024]FIG. 13 represents the DNA sequence of BVH-P4 gene from serotypeM6 S. pyogenes strain SPY67; SEQ ID NO: 13.

[0025]FIG. 14 represents the amino acid sequence BVH-P4 polypeptide fromserotype M3 S. pyogenes strain SPY67; SEQ ID NO: 14.

[0026]FIG. 15 represents the DNA sequence of BVH-P4 gene from serotypeS. pyogenes strain B514; SEQ ID NO: 15.

[0027]FIG. 16 represents the amino acid sequence BVH-P4 polypeptide fromserotype S. pyogenes strain B514; SEQ ID NO: 16.

[0028]FIG. 17 depicts the comparison of the nucleotide sequences of theBVH-P4 genes from the S. pyogens serotype M1 ATCC700294, serotype M3ATCC12384, serotype M6 SPY77 strains and the mouse isolate B514 by usingthe program Clustal W from MacVector sequence analysis software (version6.5). Identical nucleotides are presented as * and differences areindicated by blank spaces.

[0029]FIG. 18 depicts the comparison of the predicted amino acidsequences of the BVH-P4 partial open reading frames from the S. pyogenesserotype M1 ATCC700294, serotype M3 ATCC12384, serotype M6 SPY77 strainsand the mouse isolate B514 by using the program Clustal W from MacVectorsequence analysis software (version 6.5). Underneath the alignment,there is a consensus line.

[0030] Identical amino acid are illustrated with a * while differencesare indicated by periods.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides purified and isolated DNAmolecules, which encode Streptococcal polypeptides that can be used toprevent, treat, and/or diagnose Streptococcal infection.

[0032] Those skilled in the art will appreciate that the inventionincludes DNA molecules that encode analogs such as mutants, variants,homologues and derivatives of such polypeptides, as described herein inthe present patent application. The invention also includes RNAmolecules corresponding to the DNA molecules of the invention. Inaddition to the DNA and RNA molecules, the invention includes thecorresponding polypeptides and monospecific antibodies that specificallybind to such polypeptides.

[0033] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 70%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogs or thereof.

[0034] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 80%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogs or thereof.

[0035] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 90%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogs or thereof.

[0036] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 95%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogs or thereof.

[0037] According to one aspect, the present invention provides apolynucleotide encoding a polypeptide comprising a sequence chosen fromSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16 or fragments or analogs orthereof.

[0038] According to one aspect, the present invention provides apolynucleotide encoding a polypeptide capable of generating antibodieshaving binding specificity for a polypeptide having a sequence chosenfrom SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16 or fragments or analogs orthereof.

[0039] According to one aspect, the present invention provides apolynucleotide encoding an epitope bearing portion of a polypeptidehaving a sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16or fragments or analogs or thereof.

[0040] According to one aspect, the present invention relates to epitopebearing portions of a polypeptide having a sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14 or 16 or fragments or analogs or thereof.

[0041] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 70%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0042] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 80%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0043] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 90%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0044] According to one aspect, the present invention provides anisolated polynucleotide encoding a polypeptide having at least 95%identity to a second polypeptide comprising a sequence chosen from SEQID NOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0045] According to one aspect, the present invention provides apolynucleotide encoding a polypeptide comprising a sequence chosen fromSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16 or fragments or analogs orthereof.

[0046] According to one aspect, the present invention provides apolynucleotide encoding a polypeptide capable of generating antibodieshaving binding specificity for a polypeptide having a sequence chosenfrom SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0047] According to one aspect, the present invention provides apolynucleotide encoding an epitope bearing portion of a polypeptidehaving a sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0048] According to one aspect, the present invention relates to epitopebearing portions of a polypeptide having a sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14 or 16.

[0049] In accordance with the present invention, all polynucleotidesencoding polypeptides are within the scope of the present invention.

[0050] According to one aspect, the present invention relates topolypeptides having at least 70% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16 or fragments or analogs thereof.

[0051] According to one aspect, the present invention relates topolypeptides having at least 95% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16 or fragments or analogs thereof.

[0052] According to one aspect, the present invention relates topolypeptides characterized by the amino acid sequence comprisingsequences from SEQ ID NOs: 2,4,6,8,10,12,14,16 or fragments or analogsthereof.

[0053] According to one aspect, the present invention relates topolypeptides capable of generating antibodies having binding specificityfor a polypeptide having a sequence chosen from SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16 or fragments or analogs thereof.

[0054] According to one aspect, the present invention relates to epitopebearing portions of a polypeptide having a sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogs thereof.

[0055] According to one aspect, the present invention relates topolypeptides having at least 70% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16,.

[0056] According to one aspect, the present invention relates topolypeptides having at least 95% identity to a second polypeptide havingan amino acid sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16,.

[0057] According to one aspect, the present invention relates topolypeptides characterized by the amino acid sequence comprisingsequences from SEQ ID NOs: 2,4,6,8,10,12,14,16.

[0058] According to one aspect, the present invention relates topolypeptides capable of generating antibodies having binding specificityfor a polypeptide having a sequence chosen from SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16.

[0059] According to one aspect, the present invention relates to epitopebearing portions of a polypeptide having a sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14, 16.

[0060] In a further embodiment, the polypeptides in accordance-with thepresent invention are antigenic.

[0061] In a further embodiment, the polypeptides in accordance with thepresent invention are immunogenic.

[0062] In a further embodiment, the polypeptides in accordance with thepresent invention can elicit an immune response in a host.

[0063] In a further embodiment, the present invention also relates topolypeptides which are able to raise antibodies having bindingspecificity to the polypeptides of the present invention as definedabove.

[0064] An antibody that “has binding specificity” is an antibody thatrecognizes and binds the selected polypeptide but which does notsubstantially recognize and bind other molecules in a sample, e.g., abiological sample, which naturally includes the selected peptide.Specific binding can be measured using an ELISA assay in which theselected polypeptide is used as an antigen.

[0065] In accordance with the present invention, “protection” in thebiological studies is defined by a significant increase in the survivalcurve, rate or period. Statistical analysis using the Log rank test tocompare survival curves, and Fisher exact test to compare survival ratesand numbers of days to death, respectively, might be useful to calculateP values and determine whether the difference between the two groups isstatistically significant. P values of 0.05 are regarded as notsignificant.

[0066] In accordance with the present invention, there is provided aconsensus nucleotide sequence for BVH-P4 depicted in FIG. 17. As can beseen by the alignement, the polynucleotide encoding the polypeptide ofthe invention is well conserved. Without restricting the scope of theinvention, the following table A shows the possible modifications:Position on alignement in Possible nucleotide  74 G or T 130 C or T 253C or T 274 G or A 412 C or T 445 A or G 841 T or C 868 G or A 917 C or T

[0067] In accordance with the present invention, there is provided aconsensus amino acid sequence for BVH-P4 depicted in FIG. 18. As can beseen by the alignement, the polypeptide of the invention is wellconserved. Without restricting the scope of the invention, the followingtable B shows the possible modifications: Position on alignement inPossible amino acid 25 S or A

[0068] In an additional aspect of the invention there are providedantigenic/immunogenic fragments of the polypeptides of the invention, orof analogs thereof.

[0069] The fragments of the present invention should include one or moresuch epitopic regions or be sufficiently similar to such regions toretain their antigenic/immunogenic properties. Thus, for fragmentsaccording to the present invention the degree of identity is perhapsirrelevant, since they may be 100% identical to a particular part of apolypeptide or analog thereof as described herein. The present inventionfurther provides fragments having at least 10 contiguous amino acidresidues from the polypeptide sequences of the present invention. In oneembodiment, at least 15 contiguous amino acid residues. In oneembodiment, at least 20 contiguous amino acid residues.

[0070] The skilled person will appreciate that analogs of thepolypeptides of the invention will also find use in the context of thepresent invention, i.e. as antigenic/immunogenic material. Thus, forinstance proteins or polypeptides which include one or more additions,deletions, substitutions or the like are encompassed by the presentinvention.

[0071] These substitutions are those having a minimal influence on thesecondary structure and hydropathic nature of the polypeptide. Preferredsubstitutions are those known in the art as conserved, i.e. thesubstituted residues share physical or chemical properties such ashydrophobicity, size, charge or functional groups. These includesubstitutions such as those described by Dayhoff, M. in Atlas of ProteinSequence and Structure 5, 1978 and by Argos, P. in EMBO J. 8, 779-785,1989. For example, amino acids, either natural or unnatural, belongingto one of the following groups represent conservative changes:

[0072] ala, pro, gly, gln, asn, ser, thr, val;

[0073] cys, ser, tyr, thr;

[0074] val, ile, leu, met, ala, phe;

[0075] lys, arg, orn, his;

[0076] and phe, tyr, trp, his.

[0077] The preferred substitutions also include substitutions ofD-enantiomers for the corresponding L-amino acids.

[0078] The percentage of homology is defined as the sum of thepercentage of identity plus the percentage of similarity or conservationof amino acid type.

[0079] In an alternative approach, the analogs could be fusion proteins,incorporating moieties which render purification easier, for example byeffectively tagging the desired polypeptide. It may be necessary toremove the “tag” or it may be the case that the fusion polypeptideitself retains sufficient antigenicity to be useful.

[0080] Thus, what is important for analogs, derivatives and fragments isthat they possess at least a degree of the antigenicity/immunogenic ofthe protein or polypeptide from which they are derived.

[0081] As used herein, “fragments”, “analogs” or “derivatives” of thepolypeptides of the invention include those polypeptides in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably conserved) and which may benatural or unnatural.

[0082] In one embodiment, analogs of polypeptides of the invention willhave about 70% identity with those sequences illustrated in the figuresor fragments thereof. That is, 70% of the residues are the same. In afurther embodiment, polypeptides will have greater than 75% homology. Ina further embodiment, polypeptides will have greater than 80% homology.In a further embodiment, polypeptides will have greater than 85%homology. In a further embodiment, polypeptides will have greater than90% homology. In a further embodiment, polypeptides will have greaterthan 95% homology. In a further embodiment, polypeptides will havegreater than 99% homology. In a further embodiment, analogs ofpolypeptides of the invention will have fewer than about 20 amino acidresidue substitutions, modifications or deletions and more preferablyless than 10.

[0083] In a further embodiment, polypeptides will have greater than 70%homology. In a further embodiment, polypeptides will have greater than75% homology. In a further embodiment, polypeptides will have greaterthan 80% homology. In a further embodiment, polypeptides will havegreater than 85% homology. In a further embodiment, polypeptides willhave greater than 90% homology. In a further embodiment, polypeptideswill have greater than 95% homology. In a further embodiment,polypeptides will have greater than 99% homology. In a furtherembodiment, derivatives and analogs of polypeptides of the inventionwill have less than about 20 amino acid residue substitutions,modifications or deletions and more preferably less than 10. Preferredsubstitutions are those known in the art as conserved i.e. thesubstituted residues share physical or chemical properties such ashydrophobicity, size, charge or functional groups.

[0084] One can use a program such as the CLUSTAL program to compareamino acid sequences. This program compares amino acid sequences andfinds the optimal alignment by inserting spaces in either sequence asappropriate. It is possible to calculate amino acid identity orsimilarity (identity plus conservation of amino acid type) for anoptimal alignment. A program like BLASTx will align the longest stretchof similar sequences and assign a value to the fit. It is thus possibleto obtain a comparison where several regions of similarity are found,each having a different score. Both types of identity analysis arecontemplated in the present invention.

[0085] In an additional aspect of the invention there are providedantigenic/immunogenic fragments of the polypeptides of the invention, orof analogs thereof.

[0086] For fragments of the polypeptides described herein, or of analogsthereof, the situation is slightly different from native protein. It iswell known that it is possible to screen an antigenic polypeptide toidentify epitopic regions, i.e. those regions which are responsible forthe polypeptide's antigenicity or immunogenicity. Methods for carryingout such screening are well known in the art. Thus, the fragments of thepresent invention should include one or more such epitopic regions or besufficiently similar to such regions to retain theirantigenic/immunogenic properties. Thus, for fragments according to thepresent invention the degree of identity is perhaps irrelevant, sincethey may be 100% identical to a particular part of a polypeptide, analogas described herein.

[0087] Also included are polypeptides which have fused thereto othercompounds which alter the polypeptides biological or pharmacologicalproperties i.e. polyethylene glycol (PEG) to increase half-life; leaderor secretory amino acid sequences for ease of purification; prepro- andpro-sequences; and (poly)saccharides.

[0088] Furthermore, in those situations where amino acid regions arefound to be polymorphic, it may be desirable to vary one or moreparticular amino acids to more effectively mimic the different epitopesof the different streptococcus strains.

[0089] Moreover, the polypeptides of the present invention can bemodified by terminal —NH₂ acylation (eg. by acetylation, or thioglycolicacid amidation, terminal carboxy amidation, e.g. with ammonia ormethylamine) to provide stability, increased hydrophobicity for linkingor binding to a support or other molecule.

[0090] Also contemplated are hetero and homo polypeptide multimers ofthe polypeptide fragments and analogues. These polymeric forms include,for example, one or more polypeptides that have been cross-linked withcross-linkers such as avidin/biotin, gluteraldehyde ordimethylsuperimidate. Such polymeric forms also include polypeptidescontaining two or more tandem or inverted contiguous sequences, producedfrom multicistronic mRNAs generated by recombinant DNA technology. In afurther embodiment, the present invention also relates to chimericpolypeptides which comprise one or more polypeptides or fragments oranalogs thereof as defined in the figures of the present application.

[0091] In a further embodiment, the present invention also relates tochimeric polypeptides comprising two or more polypeptides having asequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragmentsor analogs thereof; provided that the polypeptides are linked as toformed a chimeric polypeptide.

[0092] In a further embodiment, the present invention also relates tochimeric polypeptides comprising two or more polypeptides having asequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16 providedthat the polypeptides are linked as to formed a chimeric polypeptide.

[0093] In order to achieve the formation of antigenic polymers (i.e.synthetic multimers), polypeptides may be utilized having bishaloacetylgroups, nitroarylhalides, or the like, where the reagents being specificfor thio groups. Therefore, the link between two mercapto groups of thedifferent polypeptides may be a single bond or may be composed of alinking group of at least two, typically at least four, and not morethan 16, but usually not more than about 14 carbon atoms.

[0094] In a particular embodiment, polypeptide fragments and analogs ofthe invention do not contain a starting residue, such as methionine(Met) or valine (Val).

[0095] Preferably, polypeptides will not incorporate a leader orsecretory sequence (signal sequence). The signal portion of apolypeptide of the invention may be determined according to establishedmolecular biological techniques. The polypeptide of interest may beisolated from a streptococcal culture and subsequently sequenced todetermine the initial residue of the mature protein and therefore thesequence of the mature polypeptide.

[0096] It is understood that polypeptides can be produced and/or usedwithout their start codon (methionine or valine) and/or without theirleader peptide to favor production and purification of recombinantpolypeptides. It is known that cloning genes without sequences encodingleader peptides will restrict the polypeptides to the cytoplasm of E.coli and will facilitate their recovery (Glick, B. R. and Pasternak, J.J. (1998) Manipulation of gene expression in prokaryotes. In “Molecularbiotechnology: Principles and applications of recombinant DNA”, 2ndedition, ASM Press, Washington D.C., p.109-143).

[0097] The polypeptides may be expressed with or without a leader orsecretion sequence. In the former case, the leader may be removed usingpost-translational processing (see U.S. Pat. No. 4,431,739, U.S. Pat.No. 4,425,437 and U.S. Pat. No. 4,338,397 incorporated herein byreference) or be chemically removed subsequent to purifying theexpressed polypeptide.

[0098] According to another aspect of the invention, there are alsoprovided (i) a composition of matter containing a polypeptide of theinvention, together with a carrier, diluent or adjuvant; (ii) apharmaceutical composition comprising a polypeptide of the invention anda carrier, diluent or adjuvant; (iii) a vaccine comprising a polypeptideof the invention and a carrier, diluent or adjuvant; (iv) a method forinducing an immune response against Streptococcus, in a host, byadministering to the host, an immunogenically effective amount of apolypeptide of the invention to elicit an immune response, e.g., aprotective immune response to Streptococcus; and particularly, (v) amethod for preventing and/or treating a Streptococcus infection, byadministering a prophylactic or therapeutic amount of a polypeptide ofthe invention to a host in need.

[0099] Before immunization, the polypeptides of the invention can alsobe coupled or conjugated to carrier proteins such as tetanus toxin,diphtheria toxin, hepatitis B virus surface antigen, poliomyelitis virusVP1 antigen or any other viral or bacterial toxin or antigen or anysuitable proteins to stimulate the development of a stronger immuneresponse. This coupling or conjugation can be done chemically orgenetically. A more detailed description of peptide-carrier conjugationis available in Van Regenmortel, M. H. V., Briand J. P., Muller S.,Plaué S., <<Synthetic Polypeptides as antigens>> in LaboratoryTechniques in Biochemistry and Molecular Biology, Vol.19 (ed.) Burdou,R. H. & Van Knippenberg P. H. (1988), Elsevier New York.

[0100] According to another aspect, there are provided pharmaceuticalcompositions comprising one or more Streptococcal polypeptides of theinvention in a mixture with a pharmaceutically acceptable adjuvant.Suitable adjuvants include (1) oil-in-water emulsion formulations suchas MF59™, SAF™, Ribi™; (2) Freund's complete or incomplete adjuvant; (3)salts i.e. AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄)₂, Al(OH)₃, AlPO₄, silica,kaolin; (4) saponin derivatives such as Stimulon™ or particles generatedtherefrom such as ISCOMs (immunostimulating complexes); (5) cytokinessuch as interleukins, interferons, macrophage colony stimulating factor(M-CSF), tumor necrosis factor (TNF); (6) other substances such ascarbon polynucleotides i.e. poly IC and poly AU, detoxified choleratoxin (CTB) and E. coli heat labile toxin for induction of mucosalimmunity. A more detailed description of adjuvant is available in areview by M. Z. I Khan et al. in Pharmaceutical Research, vol.11, No.1(1994) pp2-11, and also in another review by Gupta et al., in Vaccine,Vol.13, No14, pp1263-1276 (1995) and in WO 99/24578, which are hereinincorporated by reference. Preferred adjuvants include QuilA™, QS21™,Alhydrogel™ and Adjuphos™.

[0101] In a further embodiment, there is provided a method ofmanufacturing a pharmaceutical composition comprising admixing apolypeptide of the invention with a pharmaceutically acceptable diluent,excipient or adjuvant.

[0102] In a further aspect, the invention provides a method forprophylactic or therapeutic treatment of Streptopcoccal bacterialinfection in a host susceptible to Streptococcal infection comprisingadministering to a host a therapeutic or prophylactic amount of acomposition of the invention.

[0103] Pharmaceutical compositions of the invention may be administeredparenterally by injection, rapid infusion, nasopharyngeal absorption,dermoabsorption, or bucal or oral. Pharmaceutically acceptable carriersalso include tetanus toxoid.

[0104] Pharmaceutical compositions of the invention are used for thetreatment or prophylaxis of streptococcal infection and/or diseases andsymptoms mediated by streptococcal infection as described in P. R.Murray (Ed, in chief), E. J. Baron, M. A. Pfaller, F. C. Tenover and R.H. Yolken. Manual of Clinical Microbiology, ASM Press, Washington, D.C.sixth edition, 1995, 1482p which are herein incorporated by reference.In one embodiment, pharmaceutical compositions of the present inventionare used for the treatment or prophylaxis of pharyngitis, erysipelas andimpetigo, scarlet fever, and invasive diseases such as bacteremia andnecrotizing fasciitis and also toxic shock. In one embodiment,pharmaceutical compositions of the invention are used for the treatmentor prophylaxis of streptococcus infection and/or diseases and symptomsmediated by streptococcus infection, in particular group A streptococcus(S. pyogenes), group B streptococcus (GBS or S. agalactiae), S.pneumoniae, S. dysgalactiae, S. uberis, S. nocardia as well asStaphylococcus aureus. In a further embodiment, the streptococcusinfection is Streptococcus pyogenes.

[0105] In a particular embodiment, pharmaceutical compositions areadministered to those host at risk of streptococcus infection such asinfants, elderly and immunocompromised hosts.

[0106] According to a further aspect, the streptococcal polypeptides ofthe invention may be used in a kit comprising the polypeptides of theinvention for detection or diagnosis of streptococcal infection.

[0107] As used in the present application, the term “host” includemammals. In a further embodiment, the mammal is human.

[0108] Pharmaceutical compositions are preferably in unit dosage form ofabout 0.001 to 100 μg/kg (antigen/body weight) and more preferably 0.01to 10 μg/kg and most preferably 0.1 to 1 μg/kg 1 to 3 times with aninterval of about 1 to 6 week intervals between immunizations.

[0109] Pharmaceutical compositions are preferably in unit dosage form ofabout 0.1 μg to 10 mg and more preferably 1 μg to 1 mg and mostpreferably 10 to 100 μg 1 to 3 times with an interval of about 1 to 6week intervals between immunizations.

[0110] In one embodiment, polynucleotides are those illustrated in SEQID Nos: 1, 3, 5, 7, 9, 11, 13, 15 which may include the open readingframes (ORF), encoding the polypeptides of the invention.

[0111] It will be appreciated that the polynucleotide sequencesillustrated in the figures may be altered with degenerate codons yetstill encode the polypeptides of the invention. Accordingly the presentinvention further provides polynucleotides which hybridize to thepolynucleotide sequences herein above described (or the complementsequences thereof) having 50% identity between sequences. In oneembodiment, at least 70% identity between sequences. In one embodiment,at least 75% identity between sequences. In one embodiment, at least 80%identity between sequences. In one embodiment, at least 85% identitybetween sequences. In one embodiment, at least 90% identity betweensequences. In a further embodiment, polynucleotides are hybridizableunder stringent conditions i.e. having at least 95% identity. In afurther embodiment, more than 97% identity.

[0112] Suitable stringent conditions for hybridation can be readilydetermined by one of skilled in the art (see for example Sambrook etal., (1989) Molecular cloning: A Laboratory Manual,

[0113]₂nd ed, Cold Spring Harbor, N.Y.; Current Protocols in MolecularBiology, (1999) Edited by Ausubel F. M. et al., John Wiley & Sons, Inc.,N.Y.).

[0114] In a further embodiment, the present invention providespolynucleotides that hybridize under stringent conditions to either

[0115] (a) a DNA sequence encoding a polypeptide or

[0116] (b) the complement of a DNA sequence encoding a polypeptide;wherein said polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or16 or fragments or analogs thereof.

[0117] In a further embodiment, the present invention providespolynucleotides that hybridize under stringent conditions to either

[0118] (a) a DNA sequence encoding a polypeptide or

[0119] (b) the complement of a DNA sequence encoding a polypeptide;wherein said polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or16.

[0120] In a further embodiment, the present invention providespolynucleotides that hybridize under stringent conditions to either

[0121] (a) a DNA sequence encoding a polypeptide or

[0122] (b) the complement of a DNA sequence encoding a polypeptide;wherein said polypeptide comprises at least 10 contiguous amino acidresidues from a polypeptide comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14or 16 or fragments or analogs thereof.

[0123] In a further embodiment, the present invention providespolynucleotides that hybridize under stringent conditions to either

[0124] (a) a DNA sequence encoding a polypeptide or

[0125] (b) the complement of a DNA sequence encoding a polypeptide;wherein said polypeptide comprises at least 10 contiguous amino acidresidues from a polypeptide comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14or 16.

[0126] In a further embodiment, polynucleotides are those illustrated inSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 encoding polypeptides of theinvention.

[0127] As will be readily appreciated by one skilled in the art,polynucleotides include both DNA and RNA.

[0128] The present invention also includes polynucleotides complementaryto the polynucleotides described in the present application.

[0129] In a further aspect, polynucleotides encoding polypeptides of theinvention, or fragments, analogs or derivatives thereof, may be used ina DNA immunization method. That is, they can be incorporated into avector which is replicable and expressible upon injection therebyproducing the antigenic polypeptide in vivo. For example polynucleotidesmay be incorporated into a plasmid vector under the control of the CMVpromoter which is functional in eukaryotic cells. Preferably the vectoris injected intramuscularly.

[0130] According to another aspect, there is provided a process forproducing polypeptides of the invention by recombinant techniques byexpressing a polynucleotide encoding said polypeptide in a host cell andrecovering the expressed polypeptide product. Alternatively, thepolypeptides can be produced according to established synthetic chemicaltechniques i.e. solution phase or solid phase synthesis of oligopeptideswhich are ligated to produce the full polypeptide (block ligation).

[0131] General methods for obtention and evaluation of polynucleotidesand polypeptides are described in the following references: Sambrook etal, Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor,N.Y., 1989; Current Protocols in Molecular Biology, Edited by Ausubel F.M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols,from Molecular Cloning to Genetic Engineering, Edited by White B. A.,Humana Press, Totowa, N.J., 1997, 490 pages; Protein Purification,Principles and Practices, Scopes R. K., Springer-Verlag, New York, 3rdEdition, 1993, 380 pages; Current Protocols in Immunology, Edited byColigan J. E. et al., John Wiley & Sons Inc., New York.

[0132] For recombinant production, host cells are transfected withvectors which encode the polypeptide, and then cultured in a nutrientmedia modified as appropriate for activating promoters, selectingtransformants or amplifying the genes. Suitable vectors are those thatare viable and replicable in the chosen host and include chromosomal,non-chromosomal and synthetic DNA sequences e.g. bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA. The polypeptide sequence may beincorporated in the vector at the appropriate site using restrictionenzymes such that it is operably linked to an expression control regioncomprising a promoter, ribosome binding site (consensus region orShine-Dalgarno sequence), and optionally an operator (control element).One can select individual components of the expression control regionthat are appropriate for a given host and vector according toestablished molecular biology principles (Sambrook et al, MolecularCloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, N.Y., 1989;Current Protocols in Molecular Biology, Edited by Ausubel F. M. et al.,John Wiley and Sons, Inc. New York). Suitable promoters include but arenot limited to LTR or SV40 promoter, E. coli lac, tac or trp promotersand the phage lambda P_(L) promoter. Vectors will preferably incorporatean origin of replication as well as selection markers i.e. ampicilinresistance gene. Suitable bacterial vectors include pET, pQE70, pQE60,pQE-9, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 andeukaryotic vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG,pSVK3, PBPV, pMSG and pSVL. Host cells may be bacterial i.e. E. coli,Bacillus subtilis, Streptomyces; fungal i.e. Aspergillus niger,Aspergillus nidulins; yeast i.e. Saccharomyces or eukaryotic i.e. CHO,COS.

[0133] Upon expression of the polypeptide in culture, cells aretypically harvested by centrifugation then disrupted by physical orchemical means (if the expressed polypeptide is not secreted into themedia) and the resulting crude extract retained to isolate thepolypeptide of interest. Purification of the polypeptide from culturemedia or lysate may be achieved by established techniques depending onthe properties of the polypeptide i.e. using ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography and lectin chromatography. Finalpurification may be achieved using HPLC.

[0134] According to a further aspect, the streptococcal polypeptides ofthe invention may be used in a diagnostic test for streptococcusinfection, in particular Streptococcus pyogenes infection. Severaldiagnostic methods are possible, for example detecting streptococcusorganism in a biological sample, the following procedure may befollowed:

[0135] a) obtaining a biological sample from a host;

[0136] b) incubating an antibody or fragment thereof reactive with astreptococcus polypeptide of the invention with the biological sample toform a mixture; and

[0137] c) detecting specifically bound antibody or bound fragment in themixture which indicates the presence of streptococcus.

[0138] Alternatively, a method for the detection of antibody specific toa streptococcus antigen in a biological sample containing or suspectedof containing said antibody may be performed as follows:

[0139] a) obtaining a biological sample from a host;

[0140] b) incubating one or more streptococcus polypeptides of theinvention or fragments thereof with the biological sample to form amixture; and

[0141] c) detecting specifically bound antigen or bound fragment in themixture which indicates the presence of antibody specific tostreptococcus.

[0142] One of skill in the art will recognize that this diagnostic testmay take several forms, including an immunological test such as anenzyme-linked immunosorbent assay (ELISA), a radioimmunoassay or a latexagglutination assay, essentially to determine whether antibodiesspecific for the protein are present in an organism.

[0143] The DNA sequences encoding polypeptides of the invention may alsobe used to design DNA probes for use in detecting the presence ofstreptococcus in a biological sample suspected of containing suchbacteria. The detection method of this invention comprises:

[0144] a) obtaining the biological sample from a host;

[0145] b) incubating one or more DNA probes having a DNA sequenceencoding a polypeptide of the invention or fragments thereof with thebiological sample to form a mixture; and

[0146] c) detecting specifically bound DNA probe in the mixture whichindicates the presence of streptococcus bacteria.

[0147] The DNA probes of this invention may also be used for detectingcirculating streptococcus i.e. Streptococcus pyogenes nucleic acids in asample, for example using a polymerase chain reaction, as a method ofdiagnosing streptococcus infections. The probe may be synthesized usingconventional techniques and may be immobilized on a solid phase, or maybe labelled with a detectable label. A preferred DNA probe for thisapplication is an oligomer having a sequence complementary to at leastabout 6 contiguous nucleotides of the Streptococcus pyogenespolypeptides of the invention.

[0148] Another diagnostic method for the detection of streptococcus in ahost comprises:

[0149] a) labelling an antibody reactive with a polypeptide of theinvention or fragment thereof with a detectable label;

[0150] b) administering the labelled antibody or labelled fragment tothe host; and

[0151] c) detecting specifically bound labelled antibody or labelledfragment in the host which indicates the presence of streptococcus.

[0152] A further aspect of the invention is the use of the streptococcuspolypeptides of the invention as immunogens for the production ofspecific antibodies for the diagnosis and in particular the treatment ofstreptococcus infection. Suitable antibodies may be determined usingappropriate screening methods, for example by measuring the ability of aparticular antibody to passively protect against streptococcus infectionin a test model. One example of an animal model is the mouse modeldescribed in the examples herein. The antibody may be a whole antibodyor an antigen-binding fragment thereof and may belong to anyimmunoglobulin class. The antibody or fragment may be of animal origin,specifically of mammalian origin and more specifically of murine, rat orhuman origin. It may be a natural antibody or a fragment thereof, or ifdesired, a recombinant antibody or antibody fragment. The termrecombinant antibody or antibody fragment means antibody or antibodyfragment which was produced using molecular biology techniques. Theantibody or antibody fragments may be polyclonal, or preferablymonoclonal. It may be specific for a number of epitopes associated withthe Streptococcus pyogenes polypeptides but is preferably specific forone.

[0153] A further aspect of the invention is the use of the antibodiesdirected to the polypeptides of the invention for passive immunization.One could use the antibodies described in the present application.Suitable antibodies may be determined using appropriate screeningmethods, for example by measuring the ability of a particular antibodyto passively protect against streptococcal infection in a test model.One example of an animal model is the mouse model described in theexamples herein. The antibody may be a whole antibody or anantigen-binding fragment thereof and mav belong to any immunoglobulinclass. The antibody or fragment may be of animal origin, specifically ofmammalian origin and more specifically of murine, rat or human origin.It may be a natural antibody or a fragment thereof, or if desired, arecombinant antibody or antibody fragment. The term recombinant antibodyor antibody fragment means antibody or antibody fragment which wasproduced using molecular biology techniques. The antibody or antibodyfragments may be polyclonal, or preferably monoclonal. It may bespecific for a number of epitopes associated with the streptococcalpolypeptides but is preferably specific for one.

[0154] According to one aspect, the present invention provides the useof an antibody for treatment and/or prophylaxis of streptococcalinfections.

[0155] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLE 1 This Example Illustrates the Cloning and MolecularCharacteristics of BVH-P2 Gene and Corresponding Polypeptide

[0156] The coding region of S. pyogenes BVH-P2 gene (SEQ ID NO: 1) wasamplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene,LaJolla, Calif.) from genomic DNA of serotype M3 S. pyogenes strainATCC12384 using the following oligonuceotide primers that contained baseextensions for the addition of restriction sites NdeI (CATATG) and XhoI(CTCGAG): DMAR124 and DMAR125, which are presented in Table 1. PCRproducts were purified from agarose gel using a QIAquick gel extractionkit from QIAgen following the manufacturer's instructions (Chatsworth,Calif.), and digested with NdeI and XhoI (Pharmacia Canada Inc, Baied'Urfé, Canada). The pET-21b(+) vector (Novagen, Madison, Wis.) wasdigested with NdeI and XhoI and purified from agarose gel using aQIAquick gel extraction kit from QIAgen (Chatsworth, Calif.). TheNdeI-XhoI PCR products were ligated to the NdeI-XhoI pET-21b(+)expression vector. The ligated products were transformed into E. colistrain DH5 [φ80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1hsdR17(r_(K)−m_(K)+) deoR thi-1 supE44 λ⁻gyrA96 relA1] (Gibco BRL,Gaithersburg, Md.) according to the method of Simanis (Hanahan, D. DNACloning, 1985, D. M. Glover (ed), pp. 109-135). Recombinant pET-21b(+)plasmid (rpET21b(+)) containing BVH-P2 gene was purified using a QIAgenplasmid kit (Chatsworth, Calif.) and DNA insert was sequenced (Taq DyeDeoxy Terminator Cycle Sequencing kit, ABI, Foster City, Calif.). TABLE1 Oligonucleotide primers used for PCR amplifications of S. pyogenesgenes Restric- SEQ Primers tion ID Genes I.D. site Vector Sequence NoBVH-P2 DMAR124 NdeI pET21b 5′-CGGAGAGAACATATG 17 AAAAAGACATTAAC-3′BVH-P2 DMAR125 XhoI pET21b 5′-GGGCTCGAGCTGAAA 18 CAGTCCCTTAAAG-3′ BVH-P2DMAR507 BamHI pCMV- 5′-GAGCGGATCCTGAAC 19 GH AAAGTAG-3′ BVH-P2 DMAR508SalI pCMV- 5′-GGGGTCGACCTGAAA 20 GH CAGTCCCTTAAAG-3′ BVH-P3 DMAR188 NdeIpET21b 5′-GATGGGAAAGCATAT 21 GAGCCTCATTTTG-3′ BVH-P3 DMAR189 XhoI pET21b5′-GGCTCGAGTTTTGCT 22 AGACCTTCAG-3′ BVH-P4 DMAR192 NdeI pET21b5′-GGGTTCATACATATG 23 AACAAGAAATTTATTGG- 3′ BVH-P4 DMAR193 XhoI pET21b5′-GGCTCGAGTTTTTCA 24 GGAACTTTAATG-3′ BVH-P4 DMAR509 BamHI pCMV-5′-GTTTGGATCCTTGTG 25 GH GTAATCGTGG-3′ BVH-P4 DMAR510 SalI pCMV-5′-GGGTCGACTTTTTCA 26 GH GGAACTTTAATG-3′ BVH-P5 DMAR200 NdeI pET21b5′-GGTTCATTTTCATAT 27 GAACAAAAAAGTAATG- 3′ BVH-P5 DMAR201 XhoI pET21b5′-GGCTCGAGGTTTTCA 28 GGAACTGTGATGG-3′ BVH-P5 DMAR511 BamHI pCMV-5′-GCGGATCCTACCAAT 29 GH AACTCCGCTAAACA-3′ BVH-P5 DMAR512 SalI pCMV-5′-CAGGTCGACTTTTCA 30 GH GGAACTGTGATGGTTC- 3′ BVH-P6 DMAR235 NdeI pET21b5′-GGATAGTTTTCATAT 31 GAATCAAGAGATTAG-3′ BVH-P6 DMAR236 XhoI pET21b5′-CCCTCGAGATTGGTC 32 TGATTCCAACTATC-3′ BVH-P6 DMAR513 BamHI pCMV-5′-TTTGGATCCTAATCA 33 GH AGAGATTAGATATTC-3′ BVH-P6 DMAR514 SalI pCMV-5′-CCGTCGACATTGGTC 34 GH TGATTCCAACTATC-3′

[0157] It was determined that the open reading frame (ORF) which codesfor BVH-P2 contains 633-bp and encodes a 210 amino acid residuespolypeptide with a predicted pI of 6.40 and a predicted molecular massof 24,611.78 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO:2) using the Spscan software (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 22 aminoacid residues signal peptide (MKKTLTLLLALFAIGVTSSVRA), which ends with acleavage site situated between an alanine and a glutamic acid residues.

[0158] To confirm the presence by PCR amplification of BVH-P2 (SEQ IDNO:1) gene, the following 4 serologically distinct S. pyogenes strainswere used: the serotype M1 S. pyogenes strain ATCC 700294 and theserotype M3 S. pyogenes strain ATCC12384 were obtained from the AmericanType Culture Collection (Rockville, Md., USA); the serotype M6 S.pyogenes SPY67 clinical isolate was provided by the Centre de rechercheen infectiologie du Centre hospitalier de l'université Laval,Sainte-Foy; and S. pyogenes strain B514 which was initially isolatedfrom a mouse was provided by Susan Hollingshead, from University ofAlabama, Birmingham. The E. coli strain XL1-Blue MRF' was used in theseexperiments as negative control. Chromosomal DNA was isolated from eachS. pyogenes strain as previously described (Jayarao BM et al. 1991. J.Clin. Microbiol. 29:2774-2778). BVH-P2 (SEQ ID NO: 1) gene was amplifiedby PCR(Robocycler Gradient 96 Temperature cycler, Stratagene, LaJolla,Calif.) from the genomic DNA purified from the 4 S. pyogenes strains,and the control E. coli strain using the oligonucleotides primersDMAR124 and DMAR125 (Table 1). PCR was performed with 30 cycles of 45sec at 95° C., 45 sec at 50° C. and 1 min at 72° C. and a finalelongation period of 7 min at 72° C. The PCR products were sizefractionated in 1% agarose gels and were visualized by ethidium bromidestaining. The results of these PCR amplifications are presented in Table2. The analysis of the amplification products revealed that BVH-P2 (SEQID NO: 1) gene was present in the genome of all of the 4 S. pyogenesstrains tested. No such product was detected when the control E. coliDNA was submitted to identical PCR amplifications with theseoligonucleotide primers. TABLE 2 Identification of S. pyogenes genes byPCR amplification Identification by PCR amplification of Strain BVH-BVH- BVH- BVH- BVH- Identification P2 P3 P4 P5 P6 ATCC700294(M1) + + + + + ATCC12384 (M3) + + + + + SPY67 (M6) + + + + +B514* + + + + + E. coli XL1 Blue − − − − − MRF’

EXAMPLE 2 This Example Illustrates the Cloning and MolecularCharacteristics of BVH-P3 Gene and Corresponding Polypeptide

[0159] The coding region of S. pyogenes BVH-P3 gene (SEQ ID NO: 3) wasamplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene,LaJolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strainATCC700294 using the following oligos that contained base extensions forthe addition of restriction sites NdeI (CATATG) and XhoI (CTCGAG):DMAR188 and DMAR189, which are presented in Table 1. The methods usedfor cloning BVH-P3 into an expression vector and sequencing are similarto the methods described in Example 1.

[0160] It was determined that the open reading frame (ORF) which codesfor BVH-P3 contains 921-bp and encodes a 306 amino acid residuespolypeptide with a predicted pI of 5.73 and a predicted molecular massof 33,882.36 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO:4) using the Spscan sofware (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 27 aminoacid residues signal peptide (MSLILGAFLSVFLLVACSSTGTKTAKS), which endswith a cleavage site situated between a serine and an aspartic acidresidues. The BVH-P3 gene was shown to be present after PCRamplification using the oligonucleotide primers DMAR188 and DMAR189 inthe 4 serologically S. pyogenes strains tested (Table 2). The methodsused for PCR amplification of the BVH-P3 gene were similar to themethods presented in Example 1. No such product was detected when thecontrol E. coli DNA was submitted to identical PCR amplifications withthese oligonucleotide primers.

EXAMPLE 3 This Example Illustrates the Cloning and MolecularCharacteristics of BVH-P4 Gene and Corresponding Polypeptide

[0161] The coding region of S. pyogenes BVH-P4 gene (SEQ ID NO: 5) wasamplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene,LaJolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strainATCC700294 using the following oligos that contained base extensions forthe addition of restriction sites NdeI (CATATG) and XhoI (CTCGAG):DMAR192 and DMAR193, which are presented in Table 1. The methods usedfor cloning BVH-P4 into an expression vector and sequencing are similarto the methods described in Example 1.

[0162] It was determined that the open reading frame (ORF) which codesfor BVH-P4 contains 1053-bp and encodes a 350 amino acid residuespolypeptide with a predicted pI of 7.90 and a predicted molecular massof 36,392.50 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO:6) using the Spscan sofware (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 19 aminoacid residues signal peptide (MNKKFIGLGLASVAVLSLA), which ends with acleavage site situated between two alanine residues.

[0163] The BVH-P4 gene was shown to be present after PCR amplificationusing the oligonucleotide primers DMAR192 and DMAR193 in the 4serologically S. pyogenes strains tested (Table 2). The methods used forPCR amplification of the BVH-P4 gene were similar to the methodspresented in Example 1. No such product was detected when the control E.coli DNA was submitted to identical PCR amplifications with theseoligonucleotide primers.

[0164] Sequencing of aditional BVH-P4 genes from other strains confirmedthe high level of molecular conservation of this gene among S. pyogenesisolates. The respective coding region of S. pyogenes BVH-P4 gene fromstrains ATCC 12384 (SEQ ID NO: 11), SPY67 (SEQ ID NO: 13), and B514 (SEQID NO: 15) were amplified by PCR (Robocycler Gradient 96 Temperaturecycler, Stratagene, LaJolla, Calif.) from genomic DNA using theoligonucleotide primers DMAR192 and DMAR193 which are described inTable 1. PCR products were purified from agarose gel using a QIAquickgel extraction kit from QIAgen following the manufacturer's instructions(Chatsworth, Calif.) and the DNA inserts were sequenced (Taq Dye DeoxyTerminator Cycle Sequencing kit, ABI, Foster City, Calif.). Thepredicted amino acid sequences from strains ATCC12384 (SEQ ID NO: 12),SPY67 (SEQ ID NO: 14), and p514 (SEQ ID NO: 16) were respectivelypresented in the following FIGS. 12, 14, and 16. The FIG. 18 depicts theconsensus predicted amino acid sequences established for S. pyogenesBVH-P4. Pairwise comparison of these BVH-P4 amino acid sequencesindicated that the level of identity was higher than 99% clearly showingthe high level of conservation of BVH-P4 among S. pyogenes isolates.

EXAMPLE 4 This Example Illustrates the Cloning and MolecularCharacteristics of BVH-P5 Gene and Corresponding Polypeptide

[0165] The coding region of S. pyogenes BVH-P5 gene (SEQ ID NO: 7) wasamplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene,LaJolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strainATCC700294 using the following oligos that contained base extensions forthe addition of restriction sites NdeI (CATATG) and XhoI (CTCGAG):DMAR200 and DMAR201, which are presented in Table 1. The methods usedfor cloning BVH-P5 into an expression vector and sequencing are similarto the methods described in Example 1.

[0166] It was determined that the open reading frame (ORF) which codesfor BVH-P5 contains 1044-bp and encodes a 347 amino acid residuespolypeptide with a predicted pI of 5.65 and a predicted molecular massof 36,808.91 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO:8) using the Spscan sofware (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 17 aminoacid residues signal peptide (MNKKVMSLGLVSTALFT), which ends with acleavage site situated between a threonine and a leucine residues.

[0167] The BVH-P5 gene was shown to be present after PCR amplificationusing the oligonucleotide primers DMAR200 and DMAR201 in the 4serologically S. pyogenes strains tested,(Table 2). The methods used forPCR amplification of the BVH-P5 gene were similar to the methodspresented in example 1. No such product was detected when the control E.coli DNA was submitted to identical PCR amplifications with theseoligonucleotide primers.

EXAMPLE 5 This Example Illustrates the Cloning and MolecularCharacteristics of BVH-P6 Gene and Corresponding Polypeptide

[0168] The coding region of S. pyogenes BVH-P6 gene (SEQ ID NO:9) wasamplified by PCR (Robocycler Gradient 96 Temperature cycler, Stratagene,LaJolla, Calif.) from genomic DNA of serotype M1 S. pyogenes strainATCC700294 using the following oligonucleotide primers that containedbase extensions for the addition of restriction sites NdeI (CATATG) andXhoI (CTCGAG): DMAR235 and DMAR236, which are presented in Table 1. Themethods used for cloning BVH-P6 into an expression vector and sequencingare similar to the methods described in Example 1.

[0169] It was determined that the open reading frame (ORF) which codesfor BVH-P6 contains 1020-bp and encodes a 339 amino acid residuespolypeptide with a predicted pI of 6.66 and a predicted molecular massof 38,017.78 Da. Analysis of the predicted amino acid residues sequence(SEQ ID NO:10) using the Spscan sofware (Wisconsin Sequence AnalysisPackage; Genetics Computer Group) suggested the existence of a 33 aminoacid residues signal peptide (MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFS), whichends with a cleavage site situated between a serine and an alanineresidues. The BVH-P6 gene was shown to be present after PCRamplification using the oligonucleotide primers DMAR235 and DMAR236 inthe 4 serologically S. pyogenes strains tested, (Table 2). The methodsused for PCR amplification of the BVH-P6 gene were similar to themethods presented in example 1. No such product was detected when thecontrol E. coli DNA was submitted to identical PCR amplifications withthese oligonucleotide primers.

EXAMPLE 6 This Example Illustrates the Cloning of S. Pyogenes Genes inCMV Plasmid pCMV-GH

[0170] The DNA coding regions of S. pyogenes proteins were inserted inphase downstream of a human growth hormone (hGH) gene which was underthe transcriptional control of the cytomegalovirus (CMV) promotor in theplasmid vector PCMV-GH (Tang et al., Nature, 1992, 356 :152). The CMVpromotor is a non functional plasmid in E. coli cells but active uponadministration of the plasmid in eukaryotic cells. The vector alsoincorporated the ampicillin resistance gene.

[0171] The coding regions of BVH-P2 (SEQ ID NO: 1), BVH-P4 (SEQ ID NO:5), BVH-P5 (SEQ ID NO: 7), and BVH-P6 (SEQ ID NO: 9) genes without theirleader peptide regions were amplified by PCR (Robocycler Gradient 96Temperature cycler, Stratagene, LaJolla, Calif.) from from genomic DNAof serotype M1 S. pyogenes strain ATCC700294 using oligonucleotideprimers that contained base extensions for the addition of restrictionsites BamHI (GGATCC) and SalI (GTCGAC) which are described in Table 1.The PCR products were purified from agarose gel using a QIAquick gelextraction kit from QIAgen (Chatsworth, Calif.), digested withrestriction enzymes (Pharmacia Canada Inc, Baie d'Urfe, Canada). ThepCMV-GH vector (Laboratory of Dr. Stephen A. Johnston, Department ofBiochemistry, The University of Texas, Dallas, Tex.) was digested withBamHI and SalI and purified from agarose gel using the QIAquick gelextraction kit from QIAgen (Chatsworth, Calif.). The BamHI-SalI DNAfragments were ligated to the BamHI-SalI pCMV-GH vector to create thehGH-BVH-P2, hGH-BVHP-4, hGH-BVH-P5, and hGH-BVH-P6 fusion proteins underthe control of the CMV promoter. The ligated products were transformedinto E. coli strain DH5α [φ8dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1hsdR17(r_(K)−m_(K)+) deoR thi-1 supE44 λ⁻gyrA96 relA1] (Gibco BRL,Gaithersburg, Md.) according to the method of Simanis (Hanahan, D. DNACloning, 1985, D. M. Glover (ed), pp. 109-135). The recombinant pCMVplasmids were purified using a QIAgen plasmid kit (Chatsworth, Calif.)and the nucleotide sequences of the DNA inserts were verified by DNAsequencing.

EXAMPLE 7 This Example Illustrates the use of DNA to Elicit an ImmuneResponse to S. Pyogenes Protein Antigens

[0172] Groups of 8 female BALB/c mice (Charles River, St-Constant,Québec, Canada) were immunized by intramuscular injection of 100 μlthree times at two- or three-week intervals with 50 μg of recombinantpCMV-GH encoding BVH-P2 (SEQ ID NO: 1), BVH-P4 (SEQ ID NO: 5), BVH-P5(SEQ ID NO: 7), and BVH-P6 (SEQ ID NO: 9) genes in presence of 50 μg ofgranulocyte-macrophage colony-stimulating factor (GM-CSF)-expressingplasmid pCMV-GH-GM-CSF (Laboratory of Dr. Stephen A. Johnston,Department of Biochemistry, The University of Texas, Dallas, Tex.). Ascontrol, groups of mice were injected with 50 μg of pCMV-GH in presenceof 50 μg of pCMV-GH-GM-CSF. Blood samples were collected from theorbital sinus prior to each immunization and seven days following thethird injection and serum antibody responses were determined by ELISAusing the corresponding His-tagged labeled S. pyogenes recombinantproteins as coating antigens. The production and purification of theseHis-tagged labeled S. pyogenes recombinant proteins are presented inExample 8.

EXAMPLE 8 This Example Illustrates the Production and Purification of S.Pyogenes Recombinant Proteins

[0173] The recombinant pET-21b(+)plasmids with BVH-P2 (SEQ ID NO: 1),BVH-P3 (SEQ ID NO: 3), BVH-P4 (SEQ ID NO: 5), BVH-P5 (SEQ ID NO: 7), andBVH-P6 (SEQ ID NO: 9) were used to transform by electroporation (GenePulser II apparatus, BIO-RAD Labs, Mississauga, Canada) E. coli strainBL21 (DE3) (F⁻ompT hsdS_(B) (r⁻ _(B)m⁻ _(B)) gal dcm (DE3)) (Novagen,Madison, Wis.). In this strain of E. coli, the T7 promotor controllingexpression of the recombinant protein is specifically recognized by theT7 RNA polymerase (present on the λDE3 prophage) whose gene is under thecontrol of the lac promotor which is inducible byisopropyl-β-d-thiogalactopyranoside (IPTG). The transformants BL21(DE3)/rpET were grown at 37° C. with agitation at 250 rpm in LB broth(peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing 100 μg ofcarbenicillin (Sigma-Aldrich Canada Ltd., Oakville, Canada) per ml untilthe A₆₀₀ reached a value of 0.6. In order to induce the production ofHis-tagged S. pyogenes recombinant proteins, the cells were incubatedfor 3 additional hours in the presence of IPTG at a final concentrationof 1 mM. Induced cells from a 500 ml culture were pelleted bycentrifugation and frozen at −70° C.

[0174] The purification of the recombinant proteins from the solublecytoplasmic fraction of IPTG-induced BL21(DE3)/rpET21b(+) was done byaffinity chromatography based on the properties of the HisTag sequence(6 consecutive histidine residues) to bind to divalent cations (Ni²⁺)immobilized on the HisBind metal chelation resin. Briefly, the pelletedcells obtained from a 500 mL culture induced with IPTG was resuspendedin lysis buffer (20 mM Tris, 500 mM NaCl, 10 mM imidazole, pH 7.9)containing 1 mM PMSF, sonicated and centrifuged at 12,000×g for 20 minto remove debris. The supernatant was deposited on a Ni—NTA agarosecolumn (Qiagen, Mississauga, Ontario, Canada). The His-tagged labeled S.pyogenes recombinant proteins were eluted with 250 mM imidazole-500 mMNaCl-20 mM Tris pH 7.9. The removal of the salt and imidazole from thesamples was done by dialysis against PBS at 4° C. The quantities ofrecombinant proteins obtained from the soluble fraction of E. coli wasestimated by MicroBCA (Pierce, Rockford, Ill.).

EXAMPLE 9 This Example Illustrates the Reactivity of the His-Tagged S.Pyogenes Recombinant Proteins with Human Sera and Sera Collected fromMice after Immunization with S. Pyogenes Antigenic Preparations

[0175] As shown in Table 3, all purified recombinant proteins wererecognized in immunoblots by the antibodies present in the pool ofnormal sera. It indicates that humans which are normally in contact withS. pyogenes do develop antibodies that are specific to these proteins.These particular human antibodies might be implicated in the protectionagainst S. pyogenes infection. In addition, immunoblots also revealedthat sera collected from mice immunized with S. pyogenes antigenicpreparation enriched membrane proteins which protected mice againstlethal challenge also developed antibodies that recognized BVH-P3,BVH-P4 and BVH-P5 His-tagged recombinant proteins. This result indicatesthat these proteins were present in S. pyogenes antigenic preparationthat protected mice against infection and that they induced antibodiesthat reacted with the corresponding His-tagged recombinant protein.TABLE 3 Reactivity in immunoblots of human sera and sera collected frommice after immunization with S. pyogenes antigenic preparations with S.pyogenes His-tagged fusion recombinant proteins. Purified ApparentReactivity in immunoblots recombinant molecular with protein I.D.¹weight (kDa)² Human sera³ Mouse sera⁴ BVH-P2 25 + − BVH-P3 34 + + BVH-P435 + + BVH-P5 34 + + BVH-P6 35 + −

EXAMPLE 10 This Example Illustrates the Accessibility to Antibodies ofthe S. Pyogenes BVH-P4 Polypeptide at the Surface of IntactStreptococcal Cells

[0176] Bacteria were grown in Tood Hewitt (TH) broth (DifcoLaboratories, Detroit Mich.) with 0.5% Yeast extract (DifcoLaboratories) and 0.5% peptone extract (Merck, Darmstadt, Germany) at37° C. in a 8% CO₂ atmosphere to give an OD_(490 nm) of 0.600 (˜10⁸CFU/ml). Dilutions of anti-BVH-P4 or control sera were then added andallowed to bind to the cells, which were incubated for 2 h at 4° C.Samples were washed 4 times in blocking buffer [phosphate-bufferedsaline (PBS) containing 2% bovine serum albumin (BSA)], and then 1 ml ofgoat fluorescein (FITC)-conjugated anti-mouse IgG+IgM diluted inblocking buffer was added. After an additional incubation of 60 min atroom temperature, samples were washed 4 times in blocking buffer andfixed with 0.25 % formaldehyde in PBS buffer for 18-24 h at 40° C. Cellswere washed 2 times in PBS buffer and resuspended in 500 μl of PBSbuffer. Cells were kept in the dark at 4° C. until analyzed by flowcytometry (Epics® XL; Beckman Coulter, Inc.). Flow cytometric analysisrevealed that BVH-P4-specific antibodies efficiently recognized theircorresponding surface exposed epitopes on the heterologous (ATCC12384;serotype M3) S. pyogenes strain tested. It was determined that more than90% of the 10,000 S. pyogenes cells analyzed were labeled with theantibodies present in the BVH-P4 specific anti-sera. It appears, thatthe BVH-P4 polypeptide is accessible at the surface where it can berecognized by antibodies.

EXAMPLE 11 This Example Illustrates the Protection Against Fatal S.Pyogenes Infection induced by Passive Immunization of Mice with RabbitHyper-Immune Sera

[0177] New Zealand rabbits (Charles River laboratories, St-Constant,Canada) are injected subcutaneously at multiple sites with 50 μg and 100μg of the different His-tagged S. pyogenes recombinant proteins thatwere produced and purified as described in Example 8 and adsorbed toAlhydrogel adjuvant (Superfos Biosector a/s). Rabbits are immunizedthree times at three-week intervals with the different His-tagged S.pyogenes recombinant proteins. Blood samples are collected three weeksafter the third injection. The antibodies present in the serum arepurified by precipitation using 40% saturated ammonium sulfate. Groupsof 10 female CD-1 mice (Charles River) are injected intravenously with500 μl of purified serum collected from rabbits immunized with thedifferent His-tagged S. pyogenes recombinant proteins, or rabbitsimmunized with an unrelated control recombinant protein. Eighteen hourslater the mice are challenged with approximately 2×10⁷ CFU of the type 3S. pyogenes strain ATCC12384. Samples of the S. pyogenes challengeinoculum are plated on blood agar plates to determine the CFU and toverify the challenge dose. Deaths are recorded for a period of 5 days.

EXAMPLE 12 This Example Illustrates the Protection of Mice Against FatalS. Pyogenes Infection Induced by Immunization

[0178] Groups of 8 female CD-1 mice (Charles River) are immunizedsubcutaneously three times at three-week intervals with 20 μg ofaffinity purified His-tagged S. pyogenes recombinant proteins inpresence of 10 μg of QuilA adjuvant (Cedarlane Laboratories Ltd, Hornby,Canada) or, as control, with QuilA adjuvant alone in PBS. Blood samplesare collected from the orbital sinus on day 1, 22 and 43 prior to eachimmunization and seven days (day 50) following the third injection. Twoweeks later the mice are challenged with approximately 2×10⁷ CFU of thetype 3 S. pyogenes strain ATCC12384. Samples of the S. pyogeneschallenge inoculum are plated on blood agar plates to determine the CFUand to verify the challenge dose. Deaths are recorded for a period of 14days.

1 42 1 633 DNA S. pyogenes 1 atgaaaaaga cattaacttt gctactggca ctctttgccatcggggtaac tagtagcgtc 60 agagcggagg atgaacaaag tagtacacaa aagccagtaaaatttgattt ggatggacct 120 caacaaaaaa ttaaagatta tagtggcaac acaatcactctagaagactt atatgttggt 180 agtaaagtag taaaaatata tatccctcaa ggatggtgggtatatcttta cagacaatgt 240 gatcataaca gtaaagaacg aggaatttta gctagtcctattctcgaaaa aaatataaca 300 aaaacagatc cttatcgtca atattataca ggagtaccttatattcttaa cttaggagaa 360 gatcctttga agaaaggaga aaaattaact ttctcatttaaaggagaaga cggattttat 420 gtcggtagct atatctatag agactctgat actataaaaaaagaaaaaga agctgaagaa 480 gcacttcaaa aaaaggaaga ggaaaagcaa caaaaacagctagaagaaag catgctaaag 540 cagataagag aagaagacca taaaccttgg catcagcggttaagtgagag catccaagat 600 cagtggtgga actttaaggg actgtttcag tga 633 2 210PRT S. pyogenes 2 Met Lys Lys Thr Leu Thr Leu Leu Leu Ala Leu Phe AlaIle Gly Val 1 5 10 15 Thr Ser Ser Val Arg Ala Glu Asp Glu Gln Ser SerThr Gln Lys Pro 20 25 30 Val Lys Phe Asp Leu Asp Gly Pro Gln Gln Lys IleLys Asp Tyr Ser 35 40 45 Gly Asn Thr Ile Thr Leu Glu Asp Leu Tyr Val GlySer Lys Val Val 50 55 60 Lys Ile Tyr Ile Pro Gln Gly Trp Trp Val Tyr LeuTyr Arg Gln Cys 65 70 75 80 Asp His Asn Ser Lys Glu Arg Gly Ile Leu AlaSer Pro Ile Leu Glu 85 90 95 Lys Asn Ile Thr Lys Thr Asp Pro Tyr Arg GlnTyr Tyr Thr Gly Val 100 105 110 Pro Tyr Ile Leu Asn Leu Gly Glu Asp ProLeu Lys Lys Gly Glu Lys 115 120 125 Leu Thr Phe Ser Phe Lys Gly Glu AspGly Phe Tyr Val Gly Ser Tyr 130 135 140 Ile Tyr Arg Asp Ser Asp Thr IleLys Lys Glu Lys Glu Ala Glu Glu 145 150 155 160 Ala Leu Gln Lys Lys GluGlu Glu Lys Gln Gln Lys Gln Leu Glu Glu 165 170 175 Ser Met Leu Lys GlnIle Arg Glu Glu Asp His Lys Pro Trp His Gln 180 185 190 Arg Leu Ser GluSer Ile Gln Asp Gln Trp Trp Asn Phe Lys Gly Leu 195 200 205 Phe Gln 2103 921 DNA S. pyogenes 3 atgagcctca ttttgggtgc ttttttatct gtttttcttttagtagcttg ttcgtcaact 60 ggcactaaaa ctgctaagag tgataaatta aaagtcgtggcaaccaattc aattattgcc 120 gacatgacaa aagctattgc tggtgataaa atcgatctgcacagcattgt gccaatcggt 180 caagaccctc atgagtacga accattacca gaagatgttgaaaaaacaag taatgctgat 240 gtgattttct ataatggtat caatctagaa gatggcgggcaagcttggtt caccaaacta 300 gtgaaaaatg ctcaaaaaac gaaaaacaaa gattactttgccgtgtctga tggcattgat 360 gtgatttact tggaaggtgc aagcgaaaaa ggaaaagaagatccacatgc ttggttaaat 420 ctcgaaaacg gaatcattta ttcaaaaaac attgccaaacaattgattgc aaaggatcct 480 aaaaacaaag aaacttatga aaagaaccta aaagcttatgtggctaaatt ggaaaaacta 540 gacaaagaag ccaaatcaaa atttgatgct attgcagaaaataaaaaatt gattgtgact 600 agtgaaggct gcttcaagta cttttcaaaa gcttacggtgtcccatctgc ttatatctgg 660 gaaattaaca ccgaagaaga aggaacacca gatcaaatttcatcattgat tgaaaaacta 720 aaagtcatca agccatctgc gctttttgta gagtcaagtgtcgatagacg ccctatggaa 780 actgtttcta aagatagtgg tattcctatt tattctgagatctttacaga ttcaattgct 840 aaaaaaggta aacctggcga tagttattat gctatgatgaaatggaacct tgacaaaatt 900 tctgaaggtc tagcaaaata a 921 4 306 PRT S.pyogenes 4 Met Ser Leu Ile Leu Gly Ala Phe Leu Ser Val Phe Leu Leu ValAla 1 5 10 15 Cys Ser Ser Thr Gly Thr Lys Thr Ala Lys Ser Asp Lys LeuLys Val 20 25 30 Val Ala Thr Asn Ser Ile Ile Ala Asp Met Thr Lys Ala IleAla Gly 35 40 45 Asp Lys Ile Asp Leu His Ser Ile Val Pro Ile Gly Gln AspPro His 50 55 60 Glu Tyr Glu Pro Leu Pro Glu Asp Val Glu Lys Thr Ser AsnAla Asp 65 70 75 80 Val Ile Phe Tyr Asn Gly Ile Asn Leu Glu Asp Gly GlyGln Ala Trp 85 90 95 Phe Thr Lys Leu Val Lys Asn Ala Gln Lys Thr Lys AsnLys Asp Tyr 100 105 110 Phe Ala Val Ser Asp Gly Ile Asp Val Ile Tyr LeuGlu Gly Ala Ser 115 120 125 Glu Lys Gly Lys Glu Asp Pro His Ala Trp LeuAsn Leu Glu Asn Gly 130 135 140 Ile Ile Tyr Ser Lys Asn Ile Ala Lys GlnLeu Ile Ala Lys Asp Pro 145 150 155 160 Lys Asn Lys Glu Thr Tyr Glu LysAsn Leu Lys Ala Tyr Val Ala Lys 165 170 175 Leu Glu Lys Leu Asp Lys GluAla Lys Ser Lys Phe Asp Ala Ile Ala 180 185 190 Glu Asn Lys Lys Leu IleVal Thr Ser Glu Gly Cys Phe Lys Tyr Phe 195 200 205 Ser Lys Ala Tyr GlyVal Pro Ser Ala Tyr Ile Trp Glu Ile Asn Thr 210 215 220 Glu Glu Glu GlyThr Pro Asp Gln Ile Ser Ser Leu Ile Glu Lys Leu 225 230 235 240 Lys ValIle Lys Pro Ser Ala Leu Phe Val Glu Ser Ser Val Asp Arg 245 250 255 ArgPro Met Glu Thr Val Ser Lys Asp Ser Gly Ile Pro Ile Tyr Ser 260 265 270Glu Ile Phe Thr Asp Ser Ile Ala Lys Lys Gly Lys Pro Gly Asp Ser 275 280285 Tyr Tyr Ala Met Met Lys Trp Asn Leu Asp Lys Ile Ser Glu Gly Leu 290295 300 Ala Lys 305 5 1053 DNA S. pyogenes 5 atgaacaaga aatttattggtcttggttta gcgtcagtgg ctgtgctgag tttagctgct 60 tgtggtaatc gtggtgcttctaaaggtggg gcatcaggaa aaactgattt aaaagttgca 120 atggttaccg atactggtggtgtagatgac aaatcattca accaatcagc atgggaaggc 180 ctgcaatctt ggggtaaagaaatgggcctt caaaaaggaa caggtttcga ttattttcaa 240 tctacaagtg aatctgagtatgcaactaat ctcgatacag cagtttcagg agggtatcaa 300 ctgatttatg gtatcggctttgcattgaaa gatgctattg ctaaagcagc tggagataat 360 gaaggagtta agtttgttattatcgatgat attatcgaag gaaaagataa tgtagccagt 420 gttacctttg ccgaccatgaagctgcttat cttgcaggaa ttgcagctgc aaaaacaaca 480 aaaacaaaaa cagttggtttcgtgggcggt atggaaggaa ctgtcataac tcgatttgaa 540 aaaggttttg aagcaggagttaagtctgtt gacgatacaa tccaagttaa agttgattat 600 gctggatcat ttggtgacgctgcaaaagga aaaacaatcg cagcagctca gtatgcagca 660 ggtgctgatg ttatttaccaggcagcagga ggcactggag caggtgtatt taatgaagca 720 aaagctatta atgaaaaacgtagtgaagct gataaagttt gggttattgg tgttgaccgt 780 gatcaaaaag acgaaggaaaatacacttct aaagatggca aagaagcaaa ctttgtactt 840 gcatcatcaa tcaaagaagtcggtaaagct gttcagttaa tcaacaagca agtagcagat 900 aaaaaattcc ctggaggaaaaacaactgtc tatggtctaa aagatggcgg tgttgaaatc 960 gcaactacaa atgtttcaaaagaagctgtt aaagctatta aagaagcgaa agcaaaaatt 1020 aaatctggtg acattaaagttcctgaaaaa tag 1053 6 344 PRT S. pyogenes 6 Asn Lys Lys Phe Ile Gly LeuGly Leu Ala Ser Val Ala Val Leu Ser 1 5 10 15 Leu Ala Ala Cys Gly AsnArg Gly Ala Ser Lys Gly Gly Ala Ser Gly 20 25 30 Lys Thr Asp Leu Lys ValAla Met Val Thr Asp Thr Gly Gly Val Asp 35 40 45 Asp Lys Ser Phe Asn GlnSer Ala Trp Glu Gly Gln Ser Trp Gly Lys 50 55 60 Glu Met Gly Leu Gln LysGly Thr Gly Phe Asp Tyr Phe Gln Ser Thr 65 70 75 80 Ser Glu Ser Glu TyrAla Thr Asn Leu Asp Thr Ala Val Ser Gly Gly 85 90 95 Tyr Gln Leu Ile TyrGly Ile Gly Phe Ala Leu Lys Asp Ala Ile Ala 100 105 110 Lys Ala Ala GlyAsp Asn Gly Val Lys Phe Val Ile Ile Asp Asp Ile 115 120 125 Ile Glu GlyLys Asp Asn Val Ala Ser Val Thr Phe Ala Asp His Glu 130 135 140 Ala AlaTyr Leu Ala Gly Ile Ala Ala Ala Lys Thr Thr Lys Thr Lys 145 150 155 160Thr Val Gly Phe Val Gly Gly Met Glu Gly Thr Val Ile Thr Arg Phe 165 170175 Glu Gly Phe Glu Ala Gly Val Lys Ser Val Asp Asp Thr Ile Gln Val 180185 190 Lys Val Asp Tyr Ala Gly Ser Phe Gly Asp Ala Ala Lys Gly Lys Thr195 200 205 Ile Ala Ala Ala Gln Tyr Ala Ala Gly Ala Asp Val Ile Tyr GlnAla 210 215 220 Ala Gly Gly Thr Gly Ala Gly Val Phe Asn Glu Ala Ala IleAsn Glu 225 230 235 240 Lys Arg Ser Glu Ala Asp Lys Val Trp Val Ile GlyVal Asp Arg Asp 245 250 255 Gln Lys Asp Glu Gly Lys Tyr Thr Ser Lys AspGly Lys Glu Ala Asn 260 265 270 Phe Val Leu Ala Ser Ser Ile Lys Glu ValGly Lys Ala Val Gln Leu 275 280 285 Ile Asn Lys Gln Val Ala Asp Lys PhePro Gly Gly Lys Thr Thr Val 290 295 300 Tyr Gly Leu Lys Asp Gly Gly ValGlu Ile Ala Thr Thr Asn Val Ser 305 310 315 320 Lys Glu Ala Val Lys AlaIle Lys Glu Ala Lys Ala Lys Ile Lys Ser 325 330 335 Gly Asp Ile Lys ValPro Glu Lys 340 7 1044 DNA S. pyogenes 7 atgaacaaaa aagtaatgtcacttggtctt gtttcgactg ccctattcac attaggaggc 60 tgtaccaata actccgctaaacaaacaact gacaattcat taaaaatcgc tatgattact 120 aatcagacgg gtattgatgacaagtcattt aaccagtcag cctgggaagg cttacaagct 180 tggggaaaag aaaataaacttgaaaaagga aaaggctatg attatttcca atcagccaat 240 gaatcagagt ttaccacaaaccttgagtca gcagtaacca atggttataa tcttgttttt 300 gggattggat ttccattacatgacgctgta gaaaaagtag ccgcaaacaa tcctgacaac 360 cattttgcaa ttgtggatgatgtgattaaa ggtcaaaaaa atgttgcaag tatcaccttt 420 tcagaccatg aagcggcatacctagccggt gttgcagcag ctaaaacgac aaaaaccaag 480 caagttggtt ttgtaggtggtatggaagga gatgttgtca agcgctttga aaaaggtttt 540 gaagctggtg tgaaatcagtagatgatacc atcaaagtaa gagttgctta tgcaggctct 600 tttgcagatg ctgccaaaggcaagacgatt gcagctgctc aatacgctga aggcgcagat 660 gttatttatc atgcagcaggaggcacaggg gcgggtgtct ttagcgaagc taagtctatc 720 aacgaaaaac gtaaagaagaagataaggtt tgggttattg gtgttgaccg tgaccaaagt 780 gaagatggaa aatacactacaaaagatggc aagtcagcta attttgtttt gacctcaagt 840 atcaaggaag tcggaaaagctttagtaaaa gtagccgtaa aaacctcaga agaccaattc 900 ccaggtggtc aaataaccacttttggttta aaagaaggtg gtgttagcct tacaacggat 960 gctctgacac aagacactaaaaaagctatt gaggctgcta aaaaagcgat tatcgaagga 1020 accatcacag ttcctgaaaactaa 1044 8 347 PRT S. pyogenes 8 Met Asn Lys Lys Val Met Ser Leu GlyLeu Val Ser Thr Ala Leu Phe 1 5 10 15 Thr Leu Gly Gly Cys Thr Asn AsnSer Ala Lys Gln Thr Thr Asp Asn 20 25 30 Ser Leu Lys Ile Ala Met Ile ThrAsn Gln Thr Gly Ile Asp Asp Lys 35 40 45 Ser Phe Asn Gln Ser Ala Trp GluGly Leu Gln Ala Trp Gly Lys Glu 50 55 60 Asn Lys Leu Glu Lys Gly Lys GlyTyr Asp Tyr Phe Gln Ser Ala Asn 65 70 75 80 Glu Ser Glu Phe Thr Thr AsnLeu Glu Ser Ala Val Thr Asn Gly Tyr 85 90 95 Asn Leu Val Phe Gly Ile GlyPhe Pro Leu His Asp Ala Val Glu Lys 100 105 110 Val Ala Ala Asn Asn ProAsp Asn His Phe Ala Ile Val Asp Asp Val 115 120 125 Ile Lys Gly Gln LysAsn Val Ala Ser Ile Thr Phe Ser Asp His Glu 130 135 140 Ala Ala Tyr LeuAla Gly Val Ala Ala Ala Lys Thr Thr Lys Thr Lys 145 150 155 160 Gln ValGly Phe Val Gly Gly Met Glu Gly Asp Val Val Lys Arg Phe 165 170 175 GluLys Gly Phe Glu Ala Gly Val Lys Ser Val Asp Asp Thr Ile Lys 180 185 190Val Arg Val Ala Tyr Ala Gly Ser Phe Ala Asp Ala Ala Lys Gly Lys 195 200205 Thr Ile Ala Ala Ala Gln Tyr Ala Glu Gly Ala Asp Val Ile Tyr His 210215 220 Ala Ala Gly Gly Thr Gly Ala Gly Val Phe Ser Glu Ala Lys Ser Ile225 230 235 240 Asn Glu Lys Arg Lys Glu Glu Asp Lys Val Trp Val Ile GlyVal Asp 245 250 255 Arg Asp Gln Ser Glu Asp Gly Lys Tyr Thr Thr Lys AspGly Lys Ser 260 265 270 Ala Asn Phe Val Leu Thr Ser Ser Ile Lys Glu ValGly Lys Ala Leu 275 280 285 Val Lys Val Ala Val Lys Thr Ser Glu Asp GlnPhe Pro Gly Gly Gln 290 295 300 Ile Thr Thr Phe Gly Leu Lys Glu Gly GlyVal Ser Leu Thr Thr Asp 305 310 315 320 Ala Leu Thr Gln Asp Thr Lys LysAla Ile Glu Ala Ala Lys Lys Ala 325 330 335 Ile Ile Glu Gly Thr Ile ThrVal Pro Glu Asn 340 345 9 1020 DNA S. pyogenes 9 atgagaaaaa gatgctattcaacttcagct gcagtattgg cagcagtgac tttatttgtt 60 ctatcggtag atcgtggtgttatagcagat agtttttctg ctaatcaaga gattagatat 120 tcggaagtaa caccttatcacgttacttcc gtttggacca aaggagttac tcctccagca 180 aacttcactc aaggtgaagatgtttttcac gctccttatg ttgctaacca aggatggtat 240 gatattacca aaacattcaatggaaaagac gatcttcttt gcggggctgc cacagcaggg 300 aatatgcttc actggtggttcgatcaaaac aaagaccaaa ttaaacgtta tttggaagag 360 catccagaaa agcaaaaaataaacttcaat ggcgaacaga tgtttgacgt aaaagaagct 420 atcgacacta aaaaccaccagctagatagt aaattatttg aatattttaa agaaaaagct 480 ttcccttatc tatctactaaacacctagga gttttccctg atcatgtaat tgatatgttc 540 attaacggct accgccttagtctaactaac cacggtccaa cgccagtaaa agaaggtagt 600 aaagatcccc gaggtggtatttttgacgcc gtatttacaa gaggtgatca aagtaagcta 660 ttgacaagtc gtcatgattttaaagaaaaa aatctcaaag aaatcagtga tctcattaag 720 aaagagttaa ccgaaggcaaggctctaggc ctatcacaca cctacgctaa cgtacgcatc 780 aaccatgtta taaacctgtggggagctgac tttgattcta acgggaacct taaagctatt 840 tatgtaacag actctgatagtaatgcatct attggtatga agaaatactt tgttggtgtt 900 aattccgctg gaaaagtagctatttctgct aaagaaataa aagaagataa tattggtgct 960 caagtactag ggttatttacactttcaaca gggcaagata gttggaatca gaccaattaa 1020 10 339 PRT S. pyogenes10 Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val 1 510 15 Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 2025 30 Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 3540 45 Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 5055 60 Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr 6570 75 80 Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala85 90 95 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp100 105 110 Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys IleAsn 115 120 125 Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile AspThr Lys 130 135 140 Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe LysGlu Lys Ala 145 150 155 160 Phe Pro Tyr Leu Ser Thr Lys His Leu Gly ValPhe Pro Asp His Val 165 170 175 Ile Asp Met Phe Ile Asn Gly Tyr Arg LeuSer Leu Thr Asn His Gly 180 185 190 Pro Thr Pro Val Lys Glu Gly Ser LysAsp Pro Arg Gly Gly Ile Phe 195 200 205 Asp Ala Val Phe Thr Arg Gly AspGln Ser Lys Leu Leu Thr Ser Arg 210 215 220 His Asp Phe Lys Glu Lys AsnLeu Lys Glu Ile Ser Asp Leu Ile Lys 225 230 235 240 Lys Glu Leu Thr GluGly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala 245 250 255 Asn Val Arg IleAsn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260 265 270 Ser Asn GlyAsn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275 280 285 Ala SerIle Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290 295 300 LysVal Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala 305 310 315320 Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn 325330 335 Gln Thr Asn 11 976 DNA S. pyogenes 11 cttggtttag cgtcagtggctgtgctgagt ttagctgctt gtggtaatcg tggtgcttca 60 aaggtggggc atcaggaaaaactgatttaa aagttgcaat ggttaccgat actggtgggt 120 agatgacaaa tcattcaaccaatcagcatg ggaaggcctg caatcttggg gtaaagaatg 180 ggccttcaaa aaggaacaggtttcgattat tttcaatcta caagtgaatc tgagtagcaa 240 ctaatcttga tacagcagtttcaggagggt atcaactgat ttatggtatc ggcttgcatt 300 gaaagatgct attgctaaagcagctggaga taatgaagga gttaagtttg ttatatcgat 360 gatattatcg aaggaaaagataatgtagcc agtgttacct ttgctgacca tgagctgctt 420 atcttgcagg aattgcagctgcaaaaacaa caaaaacaaa aacagttggt ttgtgggcgg 480 tatggaagga actgtcataactcgatttga aaaaggtttt gaagcaggag taagtctgtt 540 gacgatacaa tccaagttaaagttgattat gctggatcat ttggtgacgc gcaaaaggaa 600 aaacaatcgc agcagctcagtatgcagcag gtgctgatgt tatttaccag cagcaggagg 660 cactggagca ggtgtatttaatgaagcaaa agctattaat gaaaaacgag tgaagctgat 720 aaagtttggg ttattggtgttgaccgtgat caaaaagacg aaggaaatac acttctaaag 780 atggcaaaga agcaaactttgtacttgcat catcaatcaa agaagtggta aagctgttca 840 gttaatcaac aaacaagtagcagataaaaa attccctgga ggaaaacaac tgtctatggt 900 ctaaaagatg gcggtgttgaaatcgcaact acaaatgttt caaagaagct gttaaagcta 960 ttaaagaagc gaaagc 976 12330 PRT S. pyogenes 12 Leu Gly Leu Ala Ser Val Ala Val Leu Ser Leu AlaAla Cys Gly Asn 1 5 10 15 Arg Gly Ala Ser Lys Gly Gly Ala Ser Gly LysThr Asp Leu Lys Val 20 25 30 Ala Met Val Thr Asp Thr Gly Gly Val Asp AspLys Ser Phe Asn Gln 35 40 45 Ser Ala Trp Glu Gly Leu Gln Ser Trp Gly LysGlu Met Gly Leu Gln 50 55 60 Lys Gly Thr Gly Phe Asp Tyr Phe Gln Ser ThrSer Glu Ser Glu Tyr 65 70 75 80 Ala Thr Asn Leu Asp Thr Ala Val Ser GlyGly Tyr Gln Leu Ile Tyr 85 90 95 Gly Ile Gly Phe Ala Leu Lys Asp Ala IleAla Lys Ala Ala Gly Asp 100 105 110 Asn Glu Gly Val Lys Phe Val Ile IleAsp Asp Ile Ile Glu Gly Lys 115 120 125 Asp Asn Val Ala Ser Val Thr PheAla Asp His Glu Ala Ala Tyr Leu 130 135 140 Ala Gly Ile Ala Ala Ala LysThr Thr Lys Thr Lys Thr Val Gly Phe 145 150 155 160 Val Gly Gly Met GluGly Thr Val Ile Thr Arg Phe Glu Lys Gly Phe 165 170 175 Glu Ala Gly ValLys Ser Val Asp Asp Thr Ile Gln Val Lys Val Asp 180 185 190 Tyr Ala GlySer Phe Gly Asp Ala Ala Lys Gly Lys Thr Ile Ala Ala 195 200 205 Ala GlnTyr Ala Ala Gly Ala Asp Val Ile Tyr Gln Ala Ala Gly Gly 210 215 220 ThrGly Ala Gly Val Phe Asn Glu Ala Lys Ala Ile Asn Glu Lys Arg 225 230 235240 Ser Glu Ala Asp Lys Val Trp Val Ile Gly Val Asp Arg Asp Gln Lys 245250 255 Asp Glu Gly Lys Tyr Thr Ser Lys Asp Gly Lys Glu Ala Asn Phe Val260 265 270 Leu Ala Ser Ser Ile Lys Glu Val Gly Lys Ala Val Gln Leu IleAsn 275 280 285 Lys Gln Val Ala Asp Lys Lys Phe Pro Gly Gly Lys Thr ThrVal Tyr 290 295 300 Gly Leu Lys Asp Gly Gly Val Glu Ile Ala Thr Thr AsnVal Ser Lys 305 310 315 320 Glu Ala Val Lys Ala Ile Lys Glu Ala Lys 325330 13 993 DNA S. pyogenes 13 tcttggttta gcgtcagtgg ctgtgctgagtttagctgct tgtggtaatc gtggtgcttc 60 taaaggtggg gcatcaggaa aaactgatttaaaagttgca atggttaccg atactggtgg 120 tgtagatgac aaatcattca accaatcagcatgggaaggc ctgcaatctt ggggtaaaga 180 aatgggcctt caaaaaggaa caggtttcgattattttcaa tctacaagtg aatctgagta 240 tgcaactaat ctcgatacag cagtttcaggaggatatcaa ctgatttatg gtatcggctt 300 tgcattgaaa gatgctattg ctaaagcagctggagataat gaaggagtta agtttgttat 360 tatcgatgat attatcgaag gaaaagataatgtagccagt gttacctttg ccgaccatga 420 agctgcttat cttgcaggaa ttgcggctgcaaaaacaaca aaaacaaaaa cagttggttt 480 cgtgggcggt atggaaggaa ctgtcataactcgatttgaa aaaggttttg aagcaggagt 540 taagtctgtt gacgatacaa tccaagttaaagttgattat gctggatcat ttggtgacgc 600 tgcaaaagga aaaacaatcg cagcagctcagtatgcagca ggtgctgatg ttatttacca 660 ggcagcagga ggcactggag caggtgtatttaatgaagca aaagctatta atgaaaaacg 720 tagtgaagct gataaagttt gggttattggtgttgaccgt gatcaaaaag acgaaggaaa 780 atacacttct aaagatggca aagaagcaaactttgtactt gcatcatcaa tcaaagaagt 840 tggtaaagct gttcagttaa tcaacaaacaagtagcagat aaaaaattcc ctggaggaaa 900 aacaactgtc tatggtttaa aagatggcggtgttgaaatc gcaactacaa atgtttcaaa 960 agaagctgtt aaagctatta aagaagcgaaagc 993 14 330 PRT S. pyogenes 14 Leu Gly Leu Ala Ser Val Ala Val LeuSer Leu Ala Ala Cys Gly Asn 1 5 10 15 Arg Gly Ala Ser Lys Gly Gly AlaSer Gly Lys Thr Asp Leu Lys Val 20 25 30 Ala Met Val Thr Asp Thr Gly GlyVal Asp Asp Lys Ser Phe Asn Gln 35 40 45 Ser Ala Trp Glu Gly Leu Gln SerTrp Gly Lys Glu Met Gly Leu Gln 50 55 60 Lys Gly Thr Gly Phe Asp Tyr PheGln Ser Thr Ser Glu Ser Glu Tyr 65 70 75 80 Ala Thr Asn Leu Asp Thr AlaVal Ser Gly Gly Tyr Gln Leu Ile Tyr 85 90 95 Gly Ile Gly Phe Ala Leu LysAsp Ala Ile Ala Lys Ala Ala Gly Asp 100 105 110 Asn Glu Gly Val Lys PheVal Ile Ile Asp Asp Ile Ile Glu Gly Lys 115 120 125 Asp Asn Val Ala SerVal Thr Phe Ala Asp His Glu Ala Ala Tyr Leu 130 135 140 Ala Gly Ile AlaAla Ala Lys Thr Thr Lys Thr Lys Thr Val Gly Phe 145 150 155 160 Val GlyGly Met Glu Gly Thr Val Ile Thr Arg Phe Glu Lys Gly Phe 165 170 175 GluAla Gly Val Lys Ser Val Asp Asp Thr Ile Gln Val Lys Val Asp 180 185 190Tyr Ala Gly Ser Phe Gly Asp Ala Ala Lys Gly Lys Thr Ile Ala Ala 195 200205 Ala Gln Tyr Ala Ala Gly Ala Asp Val Ile Tyr Gln Ala Ala Gly Gly 210215 220 Thr Gly Ala Gly Val Phe Asn Glu Ala Lys Ala Ile Asn Glu Lys Arg225 230 235 240 Ser Glu Ala Asp Lys Val Trp Val Ile Gly Val Asp Arg AspGln Lys 245 250 255 Asp Glu Gly Lys Tyr Thr Ser Lys Asp Gly Lys Glu AlaAsn Phe Val 260 265 270 Leu Ala Ser Ser Ile Lys Glu Val Gly Lys Ala ValGln Leu Ile Asn 275 280 285 Lys Gln Val Ala Asp Lys Lys Phe Pro Gly GlyLys Thr Thr Val Tyr 290 295 300 Gly Leu Lys Asp Gly Gly Val Glu Ile AlaThr Thr Asn Val Ser Lys 305 310 315 320 Glu Ala Val Lys Ala Ile Lys GluAla Lys 325 330 15 993 DNA S. pyogenes 15 tcttggttta gcgtcagtggctgtgctgag tttagctgct tgtggtaatc gtggtgcttc 60 taaaggtggg gcagcaggaaaaactgattt aaaagttgca atggttaccg atactggtgg 120 tgtagatgat aaatcattcaaccaatcagc atgggaaggc ctgcaatctt ggggtaaaga 180 aatgggcctt caaaaaggaacaggtttcga ttattttcaa tctacaagtg aatctgagta 240 tgcaactaat ctcgatacagcagtttcagg agggtatcaa ctgatttatg gtatcggctt 300 tgcattgaaa gatgctattgctaaagcagc tggagataat gaaggagtta agtttgttat 360 tatcgatgat attatcgaaggaaaagataa tgtagccagt gttacctttg ccgaccatga 420 agctgcttat cttgcaggaattgcagctgc aaaaacaaca aaaacaaaaa cagttggttt 480 cgtgggcggt atggaaggaactgtcataac tcgatttgaa aaaggttttg aagcaggagt 540 taagtctgtt gacgatacaatccaagttaa agttgattat gctggatcat ttggtgacgc 600 tgcaaaagga aaaacaatcgcagcagctca gtatgcagca ggtgctgatg ttatttacca 660 ggcagcagga ggcactggagcaggtgtatt taatgaagca aaagctatta atgaaaaacg 720 tagtgaagct gataaagtttgggttattgg tgttgaccgt gatcaaaaag acgaaggaaa 780 atacacttct aaagatggcaaagaagcaaa ctttgtactt gcatcatcaa tcaaagaagt 840 tggtaaagct gttcagttaatcaacaagca agtagcagat aaaaaattcc ctggaggaaa 900 aacaactgtc tatggtctaaaagatggcgg tgttgaaatc gcaactacaa atgtttcaaa 960 agaagctgtt aaagctattaaagaagcgaa agc 993 16 330 PRT S. pyogenes 16 Leu Gly Leu Ala Ser Val AlaVal Leu Ser Leu Ala Ala Cys Gly Asn 1 5 10 15 Arg Gly Ala Ser Lys GlyGly Ala Ala Gly Lys Thr Asp Leu Lys Val 20 25 30 Ala Met Val Thr Asp ThrGly Gly Val Asp Asp Lys Ser Phe Asn Gln 35 40 45 Ser Ala Trp Glu Gly LeuGln Ser Trp Gly Lys Glu Met Gly Leu Gln 50 55 60 Lys Gly Thr Gly Phe AspTyr Phe Gln Ser Thr Ser Glu Ser Glu Tyr 65 70 75 80 Ala Thr Asn Leu AspThr Ala Val Ser Gly Gly Tyr Gln Leu Ile Tyr 85 90 95 Gly Ile Gly Phe AlaLeu Lys Asp Ala Ile Ala Lys Ala Ala Gly Asp 100 105 110 Asn Glu Gly ValLys Phe Val Ile Ile Asp Asp Ile Ile Glu Gly Lys 115 120 125 Asp Asn ValAla Ser Val Thr Phe Ala Asp His Glu Ala Ala Tyr Leu 130 135 140 Ala GlyIle Ala Ala Ala Lys Thr Thr Lys Thr Lys Thr Val Gly Phe 145 150 155 160Val Gly Gly Met Glu Gly Thr Val Ile Thr Arg Phe Glu Lys Gly Phe 165 170175 Glu Ala Gly Val Lys Ser Val Asp Asp Thr Ile Gln Val Lys Val Asp 180185 190 Tyr Ala Gly Ser Phe Gly Asp Ala Ala Lys Gly Lys Thr Ile Ala Ala195 200 205 Ala Gln Tyr Ala Ala Gly Ala Asp Val Ile Tyr Gln Ala Ala GlyGly 210 215 220 Thr Gly Ala Gly Val Phe Asn Glu Ala Lys Ala Ile Asn GluLys Arg 225 230 235 240 Ser Glu Ala Asp Lys Val Trp Val Ile Gly Val AspArg Asp Gln Lys 245 250 255 Asp Glu Gly Lys Tyr Thr Ser Lys Asp Gly LysGlu Ala Asn Phe Val 260 265 270 Leu Ala Ser Ser Ile Lys Glu Val Gly LysAla Val Gln Leu Ile Asn 275 280 285 Lys Gln Val Ala Asp Lys Lys Phe ProGly Gly Lys Thr Thr Val Tyr 290 295 300 Gly Leu Lys Asp Gly Gly Val GluIle Ala Thr Thr Asn Val Ser Lys 305 310 315 320 Glu Ala Val Lys Ala IleLys Glu Ala Lys 325 330 17 29 DNA Artificial Sequence primer DMAR124 17cggagagaac atatgaaaaa gacattaac 29 18 28 DNA Artificial Sequence primerDMAR125 18 gggctcgagc tgaaacagtc ccttaaag 28 19 22 DNA ArtificialSequence primer DMAR507 19 gagcggatcc tgaacaaagt ag 22 20 28 DNAArtificial Sequence primer DMAR508 20 ggggtcgacc tgaaacagtc ccttaaag 2821 28 DNA Artificial Sequence primer DMAR188 21 gatgggaaag catatgagcctcattttg 28 22 25 DNA Artificial Sequence primer DMAR189 22 ggctcgagttttgctagacc ttcag 25 23 32 DNA Artificial Sequence primer DMAR192 23gggttcatac atatgaacaa gaaatttatt gg 32 24 27 DNA Artificial Sequenceprimer DMAR193 24 ggctcgagtt tttcaggaac tttaatg 27 25 25 DNA ArtificialSequence primer DMAR509 25 gtttggatcc ttgtggtaat cgtgg 25 26 27 DNAArtificial Sequence primer DMAR510 26 gggtcgactt tttcaggaac tttaatg 2727 31 DNA Artificial Sequence primer DMAR200 27 ggttcatttt catatgaacaaaaaagtaat g 31 28 28 DNA Artificial Sequence primer DMAR201 28ggctcgaggt tttcaggaac tgtgatgg 28 29 29 DNA Artificial Sequence primerDMAR511 29 ggggatccta ccaataactc cgctaaaca 29 30 31 DNA ArtificialSequence primer DMAR512 30 caggtcgact tttcaggaac tgtgatggtt c 31 31 30DNA Artificial Sequence primer DMAR235 31 ggatagtttt catatgaatcaagagattag 30 32 29 DNA Artificial Sequence primer DMAR236 32 ccctcgagattggtctgatt ccaactatc 29 33 30 DNA Artificial Sequence primer DMAR513 33tttggatcct aatcaagaga ttagatattc 30 34 29 DNA Artificial Sequence primerDMAR514 34 ccgtcgacat tggtctgatt ccaactatc 29 35 993 DNA S. pyogenes 35tcttggttta gcgtcagtgg ctgtgctgag tttagctgct tgtggtaatc gtggtgcttc 60taaaggtggg gcatcaggaa aaactgattt aaaagttgca atggttaccg atactggtgg 120tgtagatgac aaatcattca accaatcagc atgggaaggc ctgcaatctt ggggtaaaga 180aatgggcctt caaaaaggaa caggtttcga ttattttcaa tctacaagtg aatctgagta 240tgcaactaat ctcgatacag cagtttcagg agggtatcaa ctgatttatg gtatcggctt 300tgcattgaaa gatgctattg ctaaagcagc tggagataat gaaggagtta agtttgttat 360tatcgatgat attatcgaag gaaaagataa tgtagccagt gttacctttg ccgaccatga 420agctgcttat cttgcaggaa ttgcagctgc aaaaacaaca aaaacaaaaa cagttggttt 480cgtgggcggt atggaaggaa ctgtcataac tcgatttgaa aaaggttttg aagcaggagt 540taagtctgtt gacgatacaa tccaagttaa agttgattat gctggatcat ttggtgacgc 600tgcaaaagga aaaacaatcg cagcagctca gtatgcagca ggtgctgatg ttatttacca 660ggcagcagga ggcactggag caggtgtatt taatgaagca aaagctatta atgaaaaacg 720tagtgaagct gataaagttt gggttattgg tgttgaccgt gatcaaaaag acgaaggaaa 780atacacttct aaagatggca aagaagcaaa ctttgtactt gcatcatcaa tcaaagaagt 840cggtaaagct gttcagttaa tcaacaagca agtagcagat aaaaaattcc ctggaggaaa 900aacaactgtc tatggtctaa aagatggcgg tgttgaaatc gcaactacaa atgtttcaaa 960agaagctgtt aaagctatta aagaagcgaa agc 993 36 993 DNA S. pyogenes 36tcttggttta gcgtcagtgg ctgtgctgag tttagctgct tgtggtaatc gtggtgcttc 60taaaggtggg gcatcaggaa aaactgattt aaaagttgca atggttaccg atactggtgg 120tgtagatgac aaatcattca accaatcagc atgggaaggc ctgcaatctt ggggtaaaga 180aatgggcctt caaaaaggaa caggtttcga ttattttcaa tctacaagtg aatctgagta 240tgcaactaat cttgatacag cagtttcagg agggtatcaa ctgatttatg gtatcggctt 300tgcattgaaa gatgctattg ctaaagcagc tggagataat gaaggagtta agtttgttat 360tatcgatgat attatcgaag gaaaagataa tgtagccagt gttacctttg ctgaccatga 420agctgcttat cttgcaggaa ttgcagctgc aaaaacaaca aaaacaaaaa cagttggttt 480cgtgggcggt atggaaggaa ctgtcataac tcgatttgaa aaaggttttg aagcaggagt 540taagtctgtt gacgatacaa tccaagttaa agttgattat gctggatcat ttggtgacgc 600tgcaaaagga aaaacaatcg cagcagctca gtatgcagca ggtgctgatg ttatttacca 660ggcagcagga ggcactggag caggtgtatt taatgaagca aaagctatta atgaaaaacg 720tagtgaagct gataaagttt gggttattgg tgttgaccgt gatcaaaaag acgaaggaaa 780atacacttct aaagatggca aagaagcaaa ctttgtactt gcatcatcaa tcaaagaagt 840tggtaaagct gttcagttaa tcaacaaaca agtagcagat aaaaaattcc ctggaggaaa 900aacaactgtc tatggtctaa aagatggcgg tgttgaaatc gcaactacaa atgtttcaaa 960agaagctgtt aaagctatta aagaagcgaa agc 993 37 993 DNA S. pyogenes 37tcttggttta gcgtcagtgg ctgtgctgag tttagctgct tgtggtaatc gtggtgcttc 60taaaggtggg gcatcaggaa aaactgattt aaaagttgca atggttaccg atactggtgg 120tgtagatgac aaatcattca accaatcagc atgggaaggc ctgcaatctt ggggtaaaga 180aatgggcctt caaaaaggaa caggtttcga ttattttcaa tctacaagtg aatctgagta 240tgcaactaat ctcgatacag cagtttcagg aggatatcaa ctgatttatg gtatcggctt 300tgcattgaaa gatgctattg ctaaagcagc tggagataat gaaggagtta agtttgttat 360tatcgatgat attatcgaag gaaaagataa tgtagccagt gttacctttg ccgaccatga 420agctgcttat cttgcaggaa ttgcggctgc aaaaacaaca aaaacaaaaa cagttggttt 480cgtgggcggt atggaaggaa ctgtcataac tcgatttgaa aaaggttttg aagcaggagt 540taagtctgtt gacgatacaa tccaagttaa agttgattat gctggatcat ttggtgacgc 600tgcaaaagga aaaacaatcg cagcagctca gtatgcagca ggtgctgatg ttatttacca 660ggcagcagga ggcactggag caggtgtatt taatgaagca aaagctatta atgaaaaacg 720tagtgaagct gataaagttt gggttattgg tgttgaccgt gatcaaaaag acgaaggaaa 780atacacttct aaagatggca aagaagcaaa ctttgtactt gcatcatcaa tcaaagaagt 840tggtaaagct gttcagttaa tcaacaaaca agtagcagat aaaaaattcc ctggaggaaa 900aacaactgtc tatggtttaa aagatggcgg tgttgaaatc gcaactacaa atgtttcaaa 960agaagctgtt aaagctatta aagaagcgaa agc 993 38 993 DNA Mouse 38 tcttggtttagcgtcagtgg ctgtgctgag tttagctgct tgtggtaatc gtggtgcttc 60 taaaggtggggcagcaggaa aaactgattt aaaagttgca atggttaccg atactggtgg 120 tgtagatgataaatcattca accaatcagc atgggaaggc ctgcaatctt ggggtaaaga 180 aatgggccttcaaaaaggaa caggtttcga ttattttcaa tctacaagtg aatctgagta 240 tgcaactaatctcgatacag cagtttcagg agggtatcaa ctgatttatg gtatcggctt 300 tgcattgaaagatgctattg ctaaagcagc tggagataat gaaggagtta agtttgttat 360 tatcgatgatattatcgaag gaaaagataa tgtagccagt gttacctttg ccgaccatga 420 agctgcttatcttgcaggaa ttgcagctgc aaaaacaaca aaaacaaaaa cagttggttt 480 cgtgggcggtatggaaggaa ctgtcataac tcgatttgaa aaaggttttg aagcaggagt 540 taagtctgttgacgatacaa tccaagttaa agttgattat gctggatcat ttggtgacgc 600 tgcaaaaggaaaaacaatcg cagcagctca gtatgcagca ggtgctgatg ttatttacca 660 ggcagcaggaggcactggag caggtgtatt taatgaagca aaagctatta atgaaaaacg 720 tagtgaagctgataaagttt gggttattgg tgttgaccgt gatcaaaaag acgaaggaaa 780 atacacttctaaagatggca aagaagcaaa ctttgtactt gcatcatcaa tcaaagaagt 840 tggtaaagctgttcagttaa tcaacaagca agtagcagat aaaaaattcc ctggaggaaa 900 aacaactgtctatggtctaa aagatggcgg tgttgaaatc gcaactacaa atgtttcaaa 960 agaagctgttaaagctatta aagaagcgaa agc 993 39 330 PRT S. pyogenes 39 Leu Gly Leu AlaSer Val Ala Val Leu Ser Leu Ala Ala Cys Gly Asn 1 5 10 15 Arg Gly AlaSer Lys Gly Gly Ala Ser Gly Lys Thr Asp Leu Lys Val 20 25 30 Ala Met ValThr Asp Thr Gly Gly Val Asp Asp Lys Ser Phe Asn Gln 35 40 45 Ser Ala TrpGlu Gly Leu Gln Ser Trp Gly Lys Glu Met Gly Leu Gln 50 55 60 Lys Gly ThrGly Phe Asp Tyr Phe Gln Ser Thr Ser Glu Ser Glu Tyr 65 70 75 80 Ala ThrAsn Leu Asp Thr Ala Val Ser Gly Gly Tyr Gln Leu Ile Tyr 85 90 95 Gly IleGly Phe Ala Leu Lys Asp Ala Ile Ala Lys Ala Ala Gly Asp 100 105 110 AsnGlu Gly Val Lys Phe Val Ile Ile Asp Asp Ile Ile Glu Gly Lys 115 120 125Asp Asn Val Ala Ser Val Thr Phe Ala Asp His Glu Ala Ala Tyr Leu 130 135140 Ala Gly Ile Ala Ala Ala Lys Thr Thr Lys Thr Lys Thr Val Gly Phe 145150 155 160 Val Gly Gly Met Glu Gly Thr Val Ile Thr Arg Phe Glu Lys GlyPhe 165 170 175 Glu Ala Gly Val Lys Ser Val Asp Asp Thr Ile Gln Val LysVal Asp 180 185 190 Tyr Ala Gly Ser Phe Gly Asp Ala Ala Lys Gly Lys ThrIle Ala Ala 195 200 205 Ala Gln Tyr Ala Ala Gly Ala Asp Val Ile Tyr GlnAla Ala Gly Gly 210 215 220 Thr Gly Ala Gly Val Phe Asn Glu Ala Lys AlaIle Asn Glu Lys Arg 225 230 235 240 Ser Glu Ala Asp Lys Val Trp Val IleGly Val Asp Arg Asp Gln Lys 245 250 255 Asp Glu Gly Lys Tyr Thr Ser LysAsp Gly Lys Glu Ala Asn Phe Val 260 265 270 Leu Ala Ser Ser Ile Lys GluVal Gly Lys Ala Val Gln Leu Ile Asn 275 280 285 Lys Gln Val Ala Asp LysLys Phe Pro Gly Gly Lys Thr Thr Val Tyr 290 295 300 Gly Leu Lys Asp GlyGly Val Glu Ile Ala Thr Thr Asn Val Ser Lys 305 310 315 320 Glu Ala ValLys Ala Ile Lys Glu Ala Lys 325 330 40 330 PRT S. pyogenes 40 Leu GlyLeu Ala Ser Val Ala Val Leu Ser Leu Ala Ala Cys Gly Asn 1 5 10 15 ArgGly Ala Ser Lys Gly Gly Ala Ser Gly Lys Thr Asp Leu Lys Val 20 25 30 AlaMet Val Thr Asp Thr Gly Gly Val Asp Asp Lys Ser Phe Asn Gln 35 40 45 SerAla Trp Glu Gly Leu Gln Ser Trp Gly Lys Glu Met Gly Leu Gln 50 55 60 LysGly Thr Gly Phe Asp Tyr Phe Gln Ser Thr Ser Glu Ser Glu Tyr 65 70 75 80Ala Thr Asn Leu Asp Thr Ala Val Ser Gly Gly Tyr Gln Leu Ile Tyr 85 90 95Gly Ile Gly Phe Ala Leu Lys Asp Ala Ile Ala Lys Ala Ala Gly Asp 100 105110 Asn Glu Gly Val Lys Phe Val Ile Ile Asp Asp Ile Ile Glu Gly Lys 115120 125 Asp Asn Val Ala Ser Val Thr Phe Ala Asp His Glu Ala Ala Tyr Leu130 135 140 Ala Gly Ile Ala Ala Ala Lys Thr Thr Lys Thr Lys Thr Val GlyPhe 145 150 155 160 Val Gly Gly Met Glu Gly Thr Val Ile Thr Arg Phe GluLys Gly Phe 165 170 175 Glu Ala Gly Val Lys Ser Val Asp Asp Thr Ile GlnVal Lys Val Asp 180 185 190 Tyr Ala Gly Ser Phe Gly Asp Ala Ala Lys GlyLys Thr Ile Ala Ala 195 200 205 Ala Gln Tyr Ala Ala Gly Ala Asp Val IleTyr Gln Ala Ala Gly Gly 210 215 220 Thr Gly Ala Gly Val Phe Asn Glu AlaLys Ala Ile Asn Glu Lys Arg 225 230 235 240 Ser Glu Ala Asp Lys Val TrpVal Ile Gly Val Asp Arg Asp Gln Lys 245 250 255 Asp Glu Gly Lys Tyr ThrSer Lys Asp Gly Lys Glu Ala Asn Phe Val 260 265 270 Leu Ala Ser Ser IleLys Glu Val Gly Lys Ala Val Gln Leu Ile Asn 275 280 285 Lys Gln Val AlaAsp Lys Lys Phe Pro Gly Gly Lys Thr Thr Val Tyr 290 295 300 Gly Leu LysAsp Gly Gly Val Glu Ile Ala Thr Thr Asn Val Ser Lys 305 310 315 320 GluAla Val Lys Ala Ile Lys Glu Ala Lys 325 330 41 330 PRT S. pyogenes 41Leu Gly Leu Ala Ser Val Ala Val Leu Ser Leu Ala Ala Cys Gly Asn 1 5 1015 Arg Gly Ala Ser Lys Gly Gly Ala Ser Gly Lys Thr Asp Leu Lys Val 20 2530 Ala Met Val Thr Asp Thr Gly Gly Val Asp Asp Lys Ser Phe Asn Gln 35 4045 Ser Ala Trp Glu Gly Leu Gln Ser Trp Gly Lys Glu Met Gly Leu Gln 50 5560 Lys Gly Thr Gly Phe Asp Tyr Phe Gln Ser Thr Ser Glu Ser Glu Tyr 65 7075 80 Ala Thr Asn Leu Asp Thr Ala Val Ser Gly Gly Tyr Gln Leu Ile Tyr 8590 95 Gly Ile Gly Phe Ala Leu Lys Asp Ala Ile Ala Lys Ala Ala Gly Asp100 105 110 Asn Glu Gly Val Lys Phe Val Ile Ile Asp Asp Ile Ile Glu GlyLys 115 120 125 Asp Asn Val Ala Ser Val Thr Phe Ala Asp His Glu Ala AlaTyr Leu 130 135 140 Ala Gly Ile Ala Ala Ala Lys Thr Thr Lys Thr Lys ThrVal Gly Phe 145 150 155 160 Val Gly Gly Met Glu Gly Thr Val Ile Thr ArgPhe Glu Lys Gly Phe 165 170 175 Glu Ala Gly Val Lys Ser Val Asp Asp ThrIle Gln Val Lys Val Asp 180 185 190 Tyr Ala Gly Ser Phe Gly Asp Ala AlaLys Gly Lys Thr Ile Ala Ala 195 200 205 Ala Gln Tyr Ala Ala Gly Ala AspVal Ile Tyr Gln Ala Ala Gly Gly 210 215 220 Thr Gly Ala Gly Val Phe AsnGlu Ala Lys Ala Ile Asn Glu Lys Arg 225 230 235 240 Ser Glu Ala Asp LysVal Trp Val Ile Gly Val Asp Arg Asp Gln Lys 245 250 255 Asp Glu Gly LysTyr Thr Ser Lys Asp Gly Lys Glu Ala Asn Phe Val 260 265 270 Leu Ala SerSer Ile Lys Glu Val Gly Lys Ala Val Gln Leu Ile Asn 275 280 285 Lys GlnVal Ala Asp Lys Lys Phe Pro Gly Gly Lys Thr Thr Val Tyr 290 295 300 GlyLeu Lys Asp Gly Gly Val Glu Ile Ala Thr Thr Asn Val Ser Lys 305 310 315320 Glu Ala Val Lys Ala Ile Lys Glu Ala Lys 325 330 42 330 PRT Mouse 42Leu Gly Leu Ala Ser Val Ala Val Leu Ser Leu Ala Ala Cys Gly Asn 1 5 1015 Arg Gly Ala Ser Lys Gly Gly Ala Ala Gly Lys Thr Asp Leu Lys Val 20 2530 Ala Met Val Thr Asp Thr Gly Gly Val Asp Asp Lys Ser Phe Asn Gln 35 4045 Ser Ala Trp Glu Gly Leu Gln Ser Trp Gly Lys Glu Met Gly Leu Gln 50 5560 Lys Gly Thr Gly Phe Asp Tyr Phe Gln Ser Thr Ser Glu Ser Glu Tyr 65 7075 80 Ala Thr Asn Leu Asp Thr Ala Val Ser Gly Gly Tyr Gln Leu Ile Tyr 8590 95 Gly Ile Gly Phe Ala Leu Lys Asp Ala Ile Ala Lys Ala Ala Gly Asp100 105 110 Asn Glu Gly Val Lys Phe Val Ile Ile Asp Asp Ile Ile Glu GlyLys 115 120 125 Asp Asn Val Ala Ser Val Thr Phe Ala Asp His Glu Ala AlaTyr Leu 130 135 140 Ala Gly Ile Ala Ala Ala Lys Thr Thr Lys Thr Lys ThrVal Gly Phe 145 150 155 160 Val Gly Gly Met Glu Gly Thr Val Ile Thr ArgPhe Glu Lys Gly Phe 165 170 175 Glu Ala Gly Val Lys Ser Val Asp Asp ThrIle Gln Val Lys Val Asp 180 185 190 Tyr Ala Gly Ser Phe Gly Asp Ala AlaLys Gly Lys Thr Ile Ala Ala 195 200 205 Ala Gln Tyr Ala Ala Gly Ala AspVal Ile Tyr Gln Ala Ala Gly Gly 210 215 220 Thr Gly Ala Gly Val Phe AsnGlu Ala Lys Ala Ile Asn Glu Lys Arg 225 230 235 240 Ser Glu Ala Asp LysVal Trp Val Ile Gly Val Asp Arg Asp Gln Lys 245 250 255 Asp Glu Gly LysTyr Thr Ser Lys Asp Gly Lys Glu Ala Asn Phe Val 260 265 270 Leu Ala SerSer Ile Lys Glu Val Gly Lys Ala Val Gln Leu Ile Asn 275 280 285 Lys GlnVal Ala Asp Lys Lys Phe Pro Gly Gly Lys Thr Thr Val Tyr 290 295 300 GlyLeu Lys Asp Gly Gly Val Glu Ile Ala Thr Thr Asn Val Ser Lys 305 310 315320 Glu Ala Val Lys Ala Ile Lys Glu Ala Lys 325 330

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide chosen from: (a) a polynucleotide encoding a polypeptidehaving at least 70% identity to a second polypeptide having a sequencecomprising: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments oranalogs thereof; (b) a polynucleotide encoding a polypeptide having atleast 95% identity to a second polypeptide having a sequence comprising:SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogs thereof;(c) a polynucleotide encoding a polypeptide comprising a sequence chosenfrom: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogsthereof; (d) a polynucleotide encoding a polypeptide capable ofgenerating antibodies having binding specificity for a polypeptidehaving a sequence comprising: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 orfragments or analogs thereof; (e) a polynucleotide encoding an epitopebearing portion of a polypeptide having a sequence chosen from SEQ IDNOs: 2, 4, 6, 8, 10, 12,.14, 16 or fragments or analogs thereof; (f) apolynucleotide that is complementary to a polynucleotide in (a), (b),(c), (d), or (e).
 2. An isolated polynucleotide comprising apolynucleotide chosen from: (a) a polynucleotide encoding a polypeptidehaving at least 70% identity to a second polypeptide having a sequencecomprising: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16; (b) apolynucleotide encoding a polypeptide having at least 95% identity to asecond polypeptide having a sequence comprising: SEQ ID NOs: 2, 4, 6, 8,10, 12, 14 or 16; (c) a polynucleotide encoding a polypeptide comprisinga sequence chosen from: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16; (d) apolynucleotide encoding a polypeptide capable of generating antibodieshaving binding specificity for a polypeptide having a sequencecomprising: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16; (e) apolynucleotide encoding an epitope bearing portion of a polypeptidehaving a sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16;(f) a polynucleotide that is complementary to a polynucleotide in (a),(b), (c), (d), or (e).
 3. The polynucleotide of claim 1, wherein saidpolynucleotide is DNA.
 4. The polynucleotide of claim 2, wherein saidpolynucleotide is DNA.
 5. The polynucleotide of claim 1, wherein saidpolynucleotide is RNA.
 6. The polynucleotide of claim 2, wherein saidpolynucleotide is RNA.
 7. The polynucleotide of claim 1 that hybridizesunder stringent conditions to either (a) a DNA sequence encoding apolypeptide or (b) the complement of a DNA sequence encoding apolypeptide; wherein said polypeptide comprises SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16 or fragments or analogs thereof.
 8. The polynucleotide ofclaim 2 that hybridizes under stringent conditions to either (a) a DNAsequence encoding a polypeptide or (b) the complement of a DNA sequenceencoding a polypeptide; wherein said polypeptide comprises SEQ ID NO: 2,4, 6, 8, 10, 12, 14,
 16. 9. The polynucleotide of claim 1 thathybridizes under stringent conditions to either (a) a DNA sequenceencoding a polypeptide or (b) the complement of a DNA sequence encodinga polypeptide; wherein said polypeptide comprises at least 10 contiguousamino acid residues from a polypeptide comprising SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16 or fragments or analogs thereof.
 10. The polynucleotideof claim 2 that hybridizes under stringent conditions to either (a) aDNA sequence encoding a polypeptide or (b) the complement of a DNAsequence encoding a polypeptide; wherein said polypeptide comprises atleast 10 contiguous amino acid residues from a polypeptide comprisingSEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
 16. 11. A vector comprising thepolynucleotide of claim 1, wherein said DNA is operably linked to anexpression control region.
 12. A vector comprising the polynucleotide ofclaim 2, wherein said DNA is operably linked to an expression controlregion.
 13. A host cell transfected with the vector of claim
 11. 14. Ahost cell transfected with the vector of claim
 12. 15. A process forproducing a polypeptide comprising culturing a host cell according toclaim 13 under conditions suitable for expression of said polypeptide.16. A process for producing a polypeptide comprising culturing a hostcell according to claim 14 under condition suitable for expression ofsaid polypeptide.
 17. An isolated polypeptide comprising a polypeptidechosen from: (a) a polypeptide having at least 70% identity to a secondpolypeptide having an amino acid sequence comprising: SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16 or fragments or analogs thereof; (b) a polypeptidehaving at least 95% identity to a second polypeptide having an aminoacid sequence comprising: SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 orfragments or analogs thereof; (c) a polypeptide comprising a sequencechosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments oranalogs thereof; (d) a polypeptide capable of generating antibodieshaving binding specificity for a polypeptide having a sequence chosenfrom SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogsthereof; (e) an epitope bearing portion of a polypeptide having asequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragmentsor analogs thereof; (f) the polypeptide of (a), (b), (c), (d) or (e)wherein the N-terminal Met residue is deleted; (g) the polypeptide of(a), (b), (c), (d) or (e) wherein the secretory amino acid sequence isdeleted.
 18. An isolated polypeptide comprising a polypeptide chosenfrom: (a) a polypeptide having at least 70% identity to a secondpolypeptide having an amino acid sequence comprising: SEQ ID NOs: 2, 4,6, 8, 10, 12, 14 or 16; (b) a polypeptide having at least 95% identityto a second polypeptide having an amino acid sequence comprising: SEQ IDNOs: 2, 4, 6, 8, 10, 12, 14 or 16; (c) a polypeptide comprising asequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16; (d) apolypeptide capable of generating antibodies having binding specificityfor a polypeptide having a sequence chosen from SEQ ID NOs: 2, 4, 6, 8,10, 12, 14 or 16; (e) an epitope bearing portion of a polypeptide havinga sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16; (f) thepolypeptide of (a), (b), (c), (d) or (e) wherein the N-terminal Metresidue is deleted; (g) the polypeptide of (a), (b), (c), (d) or (e)wherein the secretory amino acid sequence is deleted.
 19. A chimericpolypeptide comprising two or more polypeptides having a sequence chosenfrom SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 or fragments or analogsthereof; provided that the polypeptides are linked as to formed achimeric polypeptide.
 20. A chimeric polypeptide comprising two or morepolypeptides having a sequence chosen from SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16; provided that the polypeptides are linked as to formed achimeric polypeptide.
 21. A pharmaceutical composition comprising apolypeptide according to any one of claims 17 to 20 and apharmaceutically acceptable carrier, diluent or adjuvant.
 22. A methodfor therapeutic or prophylactic treatment of pharyngitis, erysipelas andimpetigo, scarlet fever, and invasive diseases such as bacteremia andnecrotizing fasciitis in a host susceptible to pharyngitis, erysipelasand impetigo, scarlet fever, and invasive diseases such as bacteremiaand necrotizing fasciitis and also toxic shock comprising administeringto said host a therapeutic or prophylactic amount of a compositionaccording to claim
 21. 23. A method for therapeutic or prophylactictreatment of Streptococcus pyogenes bacterial infection in a hostsusceptible to Streptococcus pyogenes infection comprising administeringto said host a therapeutic or prophylactic amount of a compositionaccording to claim
 21. 24. A method according to claim 22 wherein thehost is an animal.
 25. A method according to claim 22 wherein the hostis a human.
 26. A method for diagnostic of streptococcal bacterialinfection in a host susceptible to streptococcal infection comprisingadministering to said host the composition of claim
 21. 27. A method fordiagnostic of streptococcal infection in a host susceptible tostreptococcal infection comprising (a) obtaining a biological samplefrom a host; (b) incubating an antibody or fragment thereof reactivewith a streptococcal polypeptide of any of the claims 17 to 20 with thebiological sample to form a mixture; and (c) detecting specificallybound antibody or bound fragment in the mixture which indicates thepresence of streptococcal.
 28. A method for diagnostic of streptococcalinfection in a host susceptible to streptococcal infection comprising(a) obtaining a biological sample from a host; (b) incubating one ormore streptococcal polypeptides of any of the claims 17 to 20 orfragments thereof with the biological sample to form a mixture; and (c)detecting specifically bound antigen or bound fragment in the mixturewhich indicates the presence of antibody specific to streptococcal. 29.Use of pharmaceutical method according to claim 22 for the prophylacticor therapeutic treatment of streptococcal bacterial infection in a hostsusceptible to streptococcal infection comprising administering to saidhost a therapeutic or prophylactic amount of a composition according toclaim
 21. 30. Kit comprising a polypeptide according to any one ofclaims 17 to 20 for detection or diagnosis of streptococcal infection.